Allergen mutants

ABSTRACT

Novel recombinant allergens with multiple mutations and reduced IgE binding affinity are disclosed. The allergens are mutants of naturally occurring allergens. The overall α-carbon backbone tertiary structure is essentially preserved. Also disclosed is a method for preparing such recombinant allergens as well as uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The application claims benefit of priority under 35 U.S.C §119(e) of provisional application serial No. 60/381,440, filed May 16,2002, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to diagnosis and treatment ofallergy. More specifically the invention provides ways of obtainingmutated allergen molecules suitable for these purposes. The inventionfurthermore relates to novel recombinant allergens, which are mutants ofnaturally occurring allergens as well as their use. Also, the inventionrelates to a composition comprising a mixture of novel recombinantmutant allergens. Further, the invention relates to a method ofpreparing such recombinant mutant allergens as well as to pharmaceuticalcompositions, including vaccines, comprising the recombinant mutantallergens. In further embodiments, the present invention relates tomethods of generating immune responses in a subject, vaccination ortreatment of a subject as well as processes for preparing thecompositions of the invention.

BACKGROUND OF THE INVENTION

[0003] Genetically predisposed individuals become sensitised (allergic)to antigens originating from a variety of environmental sources, to theallergens of which the individuals are exposed. The allergic reactionoccurs when a previously sensitised individual is re-exposed to the sameor a homologous allergen. Allergic responses range from hay fever,rhinoconductivitis, rhinitis and asthma to systemic anaphylaxis anddeath in response to e.g. bee or hornet sting or insect bite. Thereaction is immediate and can be caused by a variety of atopic allergenssuch as compounds originating from grasses, trees, weeds, insects, food,drugs, chemicals and perfumes.

[0004] However, the responses do not occur when an individual is exposedto an allergen for the first time. The initial adaptive response takestime and does usually not cause any symptoms. But when antibodies and Tcells capable of reacting with the allergen have been produced, anysubsequent exposure may provoke symptoms. Thus, allergic responsesdemonstrate that the immune response itself can cause significantpathological states, which may be life threatening.

[0005] The antibodies involved in atopic allergy belong primarily toimmunoglobulins of the IgE class. IgE binds to specific receptors on thesurface of mast cells and basophils. Following complex formation of aspecific allergen with IgE bound to mast cells, receptor cross-linkingon the cell surface results in signalling through the receptors and thephysiological response of the target cells. Degranulation of a mast cellresults in the release of i.a. histamine, heparin, a chemotactic factorfor eosinophilic leukocytes, leukotrienes C4, D4 and E4, which causeprolonged constriction of the bronchial smooth muscle cells. Theresulting effects may be systemic or local in nature.

[0006] The antibody-mediated hypersensitivity reactions can be dividedinto four classes, namely type I, type II, type III and type IV. Type Iallergic reactions is the classic immediate hypersensitivity reactionoccurring within seconds or minutes following antigen exposure. Thesesymptoms are mediated by allergen specific IgE.

[0007] Commonly, allergic reactions are observed as a response toprotein allergens present e.g. in pollens, house dust mites, animal hairand dandruff, venoms, and food products.

[0008] In order to reduce or eliminate allergic reactions, carefullycontrolled and repeated administration of allergy vaccines is commonlyused. Allergy vaccination is traditionally performed by parenteral,intranasal, or sublingual administration in increasing doses over afairly long period of time, and results in desensitisation of thepatient. The exact immunological mechanism is not known, but induceddifferences in the phenotype of allergen specific T cells is thought tobe of particular importance.

[0009] Allergy Vaccination

[0010] The concept of vaccination is based on two fundamentalcharacteristics of the immune system, namely specificity and memory.Vaccination will prime the immune system of the recipient, and uponrepeated exposure to similar proteins the immune system will be in aposition to respond more rigorously to the challenge of for example amicrobial infection. Vaccines are mixtures of proteins intended to beused in vaccination for the purpose of generating such a protectiveimmune response in the recipient. The protection will comprise onlycomponents present in the vaccine and homologous antigens.

[0011] Compared to other types of vaccination allergy vaccination iscomplicated by the existence of an ongoing immune response in allergicpatients. This immune response is characterised by the presence ofallergen specific IgE mediating the release of allergic symptoms uponexposure to allergens. Thus, allergy vaccination using allergens fromnatural sources has an inherent risk of side effects being in the utmostconsequence life threatening to the patient.

[0012] Approaches to circumvent this problem may be divided in threecategories. In practise measures from more than one category are oftencombined. First category of measures includes the administration ofseveral small doses over prolonged time to reach a substantialaccumulated dose. Second category of measures includes physicalmodification of the allergens by incorporation of the allergens into gelsubstances such as aluminium hydroxide. Aluminium hydroxide formulationhas an adjuvant effect and a depot effect of slow allergen releasereducing the tissue concentration of active allergen components. Thirdcategory of measures include chemical modification of the allergens forthe purpose of reducing allergenicity, i.e. IgE binding.

[0013] The detailed mechanism behind successful allergy vaccinationremains controversial. It is, however, agreed that T cells play a keyrole in the overall regulation of immune responses. According to currentconsensus the relation between two extremes of T cell phenotypes, Th1and Th2, determine the allergic status of an individual. Uponstimulation with allergen Th1 cells secrete interleukines dominated byinterferon-y leading to protective immunity and the individual ishealthy. Th2 cells on the other hand secrete predominantly interleukin 4and 5 leading to IgE synthesis and eosinophilia and the individual isallergic. In vitro studies have indicated the possibility of alteringthe responses of allergen specific T cells by challenge with allergenderived peptides containing relevant T cell epitopes. Current approachesto new allergy vaccines are therefore largely based on addressing the Tcells, the aim being to silence the T cells (anergy induction) or toshift the response from the Th2 phenotype to the Th1 phenotype.

[0014] Antibody-Binding Epitopes (B-Cell Epitopes)

[0015] X-ray crystallographic analyses of F_(ab)-antigen complexes hasincreased the understanding of antibody-binding epitopes. According tothis type of analysis antibody-binding epitopes can be defined as asection of the surface of the antigen comprising atoms from 15-25 aminoacid residues, which are within a distance from the atoms of theantibody enabling direct interaction. The affinity of theantigen-antibody interaction can not be predicted from the enthalpycontributed by van der Waals interactions, hydrogen bonds or ionicbonds, alone. The entropy associated with the almost complete expulsionof water molecules from the interface represent an energy contributionsimilar in size. This means that perfect fit between the contours of theinteracting molecules is a principal factor underlying antigen-antibodyhigh affinity interactions.

[0016] In WO 97/30150 (ref. 1), a population of protein molecules isclaimed, which protein molecules have a distribution of specificmutations in the amino acid sequence as compared to a parent protein.From the description, it appears that the invention is concerned withproducing analogues which are modified as compared to the parentprotein, but which are taken up, digested and presented to T cells inthe same manner as the parent protein (naturally occurring allergens).Thereby, a modified T cell response is obtained. Libraries of modifiedproteins are prepared using a technique denoted PM (ParsimoniousMutagenesis).

[0017] In WO 92/02621 (ref. 2), recombinant DNA molecules are described,which molecules comprise a DNA coding for a polypeptide having at leastone epitope of an allergen of trees of the order Fagales, the allergenbeing selected from Aln g 1, Cor a 1 and Bet v 1. The recombinantmolecules described herein do all have an amino acid sequence or part ofan amino acid sequence that corresponds to the sequence of a naturallyoccurring allergen.

[0018] WO 90/11293 (ref. 3) relates i.a. to isolated allergenic peptidesof ragweed pollen and to modified ragweed pollen peptides. The peptidesdisclosed therein have an amino acid sequence corresponding either tothe sequence of the naturally occurring allergen or to naturallyoccurring isoforms thereof.

[0019] Chemical Modification of Allergens

[0020] Several approaches to chemical modification of allergens havebeen taken. Approaches of the early seventies include chemical couplingof allergens to polymers, and chemical cross-linking of allergens usingformaldehyde, etc., producing the so-called ‘allergoids’. The rationalebehind these approaches was random destruction of IgE binding epitopesby attachment of the chemical ligand thereby reducing IgE-binding whileretaining immunogenicity by the increased molecular weight of thecomplexes. Inherent disadvantages of ‘allergoid’ production are linkedto difficulties in controlling the process of chemical cross-linking anddifficulties in analysis and standardisation of the resulting highmolecular weight complexes. ‘Allergoids’ are currently in clinical useand due to the random destruction of IgE binding epitopes higher dosescan be administered as compared to conventional vaccines, but the safetyand efficacy parameters are not improved over use of conventionalvaccines.

[0021] More recent approaches to chemical modification of allergens aimat a total disruption of the tertiary structure of the allergen thuseliminating IgE binding assuming that the essential therapeutic targetis the allergen specific T cell. Such vaccines contain allergen sequencederived synthetic peptides representing minimal T cells epitopes, longerpeptides representing linked T cells epitopes, longer allergen sequencederived synthetic peptides representing regions of immunodominant T cellepitopes, or allergen molecules cut in two halves by recombinanttechnique. Another approach based on this rationale has been theproposal of the use of “low IgE binding” recombinant isoforms. In recentyears it has become clear that natural allergens are heterogeneouscontaining isoallergens and variants having up to approximately 25% oftheir amino acids substituted. Some recombinant isoallergens have beenfound to be less efficient in IgE binding possibly due to irreversibledenaturation and hence total disruption of tertiary structure.

[0022] In Vitro Mutagenesis and Allergy Vaccination

[0023] Attempts to reduce allergenicity by in vitro site directedmutagenesis have been performed using several allergens including Der f2 (Takai et al, ref. 4), Der p 2 (Smith et al, ref. 5), a 39 kDaDermatophagoides farinae allergen (Aki et al, ref. 6), bee venomphospholipase A2 (Förster et al, ref. 7), Ara h 1 (Burks et al, ref. 8),Ara h 2 (Stanley et al, ref. 9), Bet v 1 (Ferreira et al, ref. 10 and11), birch profilin (Wiedemann et al, ref. 12), and Ory s 1 (Alvarez etal, ref. 13).

[0024] The rationale behind these approaches, again, is addressingallergen specific T cells while at the same time reducing the risk ofIgE mediated side effects by reduction or elimination of IgE binding bydisruption of the tertiary structure of the recombinant mutant allergen.

[0025] The article by Ferreira et al (ref. 11) discloses the use of sitedirected mutagenesis for the purpose of reducing IgE binding. Althoughthe three-dimensional structure of Bet v 1 is mentioned in the articlethe authors do not use the structure for prediction of solvent exposedamino acid residues for mutation, half of which have a low degree ofsolvent exposure. Rather they use a method developed for prediction offunctional residues in proteins. Although the authors do discussconservation of α-carbon backbone tertiary structure this concept is nota part of the therapeutic strategy but merely included to assess invitro IgE binding. Furthermore, the evidence presented is not adequatesince normalisation of CD-spectra prevents the evaluation ofdenaturation of a proportion of the sample, which is a common problem.The therapeutic strategy described aim at inducing tolerance in allergenspecific T cells and initiation of a new immune response is notmentioned.

[0026] The article by Wiedemann et al. (ref. 12) describes the use ofsite directed mutagenesis and peptide synthesis for the purpose ofmonoclonal antibody epitope characterisation. The study demonstratesthat substitution of a surface exposed amino acid has the capacity tomodify the binding characteristics of a monoclonal antibody, which isnot surprising considering common knowledge. The experiments describedare not designed to assess modulation in the binding of polyclonalantibodies such as allergic patients' serum IgE. One of the experimentsdoes apply serum IgE and although this experiment is not suitable forquantitative assessment, IgE binding does not seem to be affected by themutations performed.

[0027] The article by Smith et al. (ref. 5) describes the use of sitedirected mutagenesis for the purpose of monoclonal antibody epitopemapping and reduction of IgE binding. The authors have no knowledge ofthe tertiary structure and make no attempt to assess the conservation ofα-carbon backbone tertiary structure. The algorithm used does not ensurethat amino acids selected for mutation are actually exposed to themolecular surface. Only one of the mutants described lead to asubstantial reduction in IgE binding. This mutant is deficient inbinding of all antibodies tested indicating that the tertiary structureis disrupted. The authors do not define a therapeutic strategy andinitiation of a new immune response is not mentioned.

[0028] The article by Colombo et al. (ref. 14) describes the study of anIgE binding epitope by use of site directed mutagenesis and peptidesynthesis. The authors use a three dimensional computer model structurebased on the crystal structure of a homologous protein to illustrate thepresence of the epitope on the molecular surface. The further presenceof an epitope on a different allergen showing primary structure homologyis addressed using synthetic peptides representing the epitope. Thetherapeutic strategy is based on treatment using this synthetic peptiderepresenting a monovalent IgE binding epitope.

[0029] The article by Spangfort et al. (ref. 15) describes thethree-dimensional structure and conserved surface exposed areas of themajor birch allergen. The article does not disclose site directedmutagenesis, neither is therapeutic application addressed.

[0030] In none of the studies described above is IgE binding beingreduced by substitution of surface exposed amino acids while conservingα-carbon backbone tertiary structure. Neither is the concept ofinitiating a new protective immune response mentioned.

[0031] WO 01/83559 discloses a method of selecting a protein variantwith modified immunogenicity by using antibody binding peptide sequencesto localise epitope sequences on the 3-dimensional structure of theparent protein. An epitope area is subsequently defined and one or moreof the amino acids defining the epitope area are mutated. The inventionis exemplified by industrial enzymes that function as allergens.

[0032] WO 99/47680 discloses the introduction of artificial amino acidsubstitutions into defined critical positions while retaining theα-carbon backbone tertiary structure of the allergen. In particular, WO99/47680 discloses a recombinant allergen, which is a non-naturallyoccurring mutant derived from a naturally occurring allergen, wherein atleast one surface-exposed, conserved amino acid residue of a B cellepitope is substituted by another residue which does not occur in thesame position in the amino acid sequence of any known homologous proteinwithin the taxonomic order from which said naturally occurring allergenoriginates, said mutant allergen having essentially the same α-carbonbackbone tertiary structure as said naturally occurring allergen, andthe specific IgE binding to the mutated allergen being reduced ascompared to the binding to said naturally occurring allergen.

[0033] The recombinant allergen disclosed in WO 99/47680 is obtainableby a) identifying amino acid residues in a naturally occurring allergenwhich are conserved with more than 70% identity in all known homologousproteins within the taxonomic order from which said naturally occurringallergen originates, b) defining at least one patch of conserved aminoacid residues being coherently connected over at least 400 Å² of thesurface of the three-dimensional structure of the allergen molecule asdefined by having a solvent accessibility of at least 20%, said at leastone patch comprising at least one B cell epitope, and c) substituting atleast one amino acid residue in said at least one patch by another aminoacid being non-conservative in the particular position while essentiallypreserving the overall α-carbon backbone tertiary structure of theallergen molecule.

[0034] Patent application PCT/DK 01/00764 relates to mutants ofnaturally occurring allergens. The following specific Bet v 1 mutantsare disclosed therein:

[0035] Mutant A: Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu,Asp156His, +160Asn.

[0036] Mutant B: Tyr5Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val,Lys123lle, Lys134Glu, Asp156His.

[0037] Mutant 2628: Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu.

[0038] Mutant 2637: Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly,Leu152Lys, Ala153Gly, Ser155Pro.

[0039] Mutant 2724: N28T, K32Q, N78K, K103V, P108G, R145E, D156H, +160N.

[0040] Mutant 2733: Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu45Ser,Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu,Asp156His, +160Asn.

[0041] Mutant 2744: Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys,Lys103Val, Lys123lle, Asp156His, +160Asn.

[0042] Mutant 2753: Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser,Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu.

[0043] Mutant 2744+2595: Y5V, N28T, K32Q, E42S, E45S, N78K, K103V,P108G, K123I, K134E, D156H, +160N.

[0044] Mutant 2744+2628: Y5V, E42S, E45S, K65N, N78K, K97S, K103V,K123I, K134E, D156H, +160N.

[0045] Mutant 2744+2595+2628: Y5V, N28T, K32Q, E42S, E45S, K65N, N78K,K97S, K103V, P108G, K123I, K134E, D156H, +160N.

BRIEF DESCRIPTION OF THE FIGURES

[0046]FIG. 1 shows a theoretical model of the reaction between anallergen and mast cells by IgE cross-linking.

[0047]FIG. 2. (Left) The molecular surface of Bet v 1 with the locationof group 1 to 10 shown in black and grey tones. (Right) View of theamino acid residues constituting group 1 to 10. Groups are marked 1 to10.

[0048]FIG. 3 shows mutant-specific oligonucleotide primers used formutation of Bet v 1 mutant number 1. Mutated nucleotides are underlined.

[0049]FIG. 4 shows oligonucleotide primers for site directed mutagenesisof Bet v 1 (No. 2801). Two generally applicable primers denoted“all-sense” and “all non-sense” were used for all mutants.

[0050]FIG. 5 shows the DNA and amino acid sequence of the naturallyoccurring allergen Bet v 1 as well as a number of Bet v 1 mutations.

[0051]FIG. 6 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant.

[0052]FIG. 7 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 mutant Asn28Thr+Lys32Gln.

[0053]FIG. 8 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Pro108Gly mutant.

[0054]FIG. 9 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu60Ser mutant.

[0055]FIG. 10 shows the CD spectra of recombinant and the (Asn28Thr,Lys32Gln, Glu45Ser, Pro108Gly) mutant, recorded at close to equalconcentrations.

[0056]FIG. 11 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by the (Asn28Thr, Lys32Gln, Glu45Ser,Pro108Gly) mutant.

[0057]FIG. 12 shows a graphical illustration of the 2-step PCR mutationtechnique used for generating mutated Bet v 1 allergens. (I) shows thesense and antisense primers. (II) shows the final recombinant proteinharbouring mutations at the indicated positions. Lines represent DNAsequences; numbers in parentheses above lines represent senseoligonucleotide primers (1), (3), (5), (7), (9) and (11); numbers inparentheses below lines represent anti-sense nucleotide primers (2), (4)(6), (8), (10) and (12); notation is X (position) Y representsmutations; (1) represents the sense oligonucleotide primer accommodatingthe proteins N-terminus; and (12) represents the anti-senseoligonucleotide accommodating the protein C-terminus.

[0058]FIG. 13 shows a graphical illustration of the PCR mutation eventsleading to the cloning of Bet v 1 (3004), (3005), (3007) and (3009).Primers used for introducing point mutations are listed.

[0059]FIG. 14 shows a graphical illustration of the PCR mutation eventsleading to the cloning of Bet v 1 (3031) to (3045). Degenerated primersused for introducing random mutations in position 10, 20, 36, 73, 87,129 and 149 are listed. The possible outcome of mutation for eachposition is shown at the top.

[0060]FIG. 15 shows schematically the primers used to create themutations. (I) shows the sense and antisense primers. (II) shows thefinal recombinant protein harbouring mutations at the indicatedpositions. Lines represent DNA sequences; numbers in parentheses abovelines represent sense oligonucleotide primers (1), (3), (5), (7), (9)and (11); numbers in parentheses below lines represent anti-sensenucleotide primers (2), (4) (6), (8), (10) and (12); notation is X(position) Y represents mutations; (1) represents the senseoligonucleotide primer accommodating the proteins N-terminus; and (12)represents the anti-sense oligonucleotide accommodating the proteinC-terminus.

[0061]FIG. 16 shows an illustration of the construction of Bet v 1mutants and a listing of the primers used. The mutants contain from fiveto nine amino acids.

[0062]FIG. 17 shows introduced point mutations at the surface of Bet v 1(2628) and Bet v 1 (2637) showing backbone+amino acids 95-100% conservedamong Fagales (grey) and introduced point mutations (black). In mutantBet v 1 (2628), five primary mutations were introduced in one half ofBet v 1 leaving the other half unaltered. In mutant Bet v 1 (2637), fiveprimary and three secondary mutations were introduced in the other half,leaving the first half unaltered.

[0063]FIG. 18 shows the circular dichroism (CD) spectra of recombinantBet v 1.2801 (wild type; closed circles) and the Bet v 1 (2637) mutant(open squares) recorded at nearly identical concentrations.

[0064]FIG. 19 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1.2801 (wild type) to serum IgE from a pool ofallergic patients by non-biotinylated Bet v 1.2801 and by Bet v 1(2628), Bet v 1 (2637), and a 1:1 mix of Bet v 1 (2628) and Bet v1(2637).

[0065]FIG. 20 shows histamine release in human basophil cells of Bet v1.2801 (wild type), Bet v 1 (2628), and Bet v 1 (2637).

[0066]FIG. 21 shows histamine release in human basophil cells of Bet v1.2801 (wild type), Bet v 1 (2628), and Bet v 1 (2637).

[0067]FIG. 22 shows point mutations at the surface of Bet v 1 (2744).Grey: Back bone+Amino acid residues 95-100% conserved among Fagales.Black: Point mutations.

[0068]FIG. 23 shows point mutations at the surface of Bet v 1 (2753).Grey: Back bone+Amino acid residues 95-100% conserved among Fagales.Black: Point mutations.

[0069]FIG. 24 shows point mutations at the surface of Bet v 1 (2744)[white] and Bet v 1 (2753) [Black]. Grey: Back bone+Amino acid residues95-100% conserved among Fagales. Black: Mutations (Y5V, K134E), (E42S,E45S), (N78K, K103V), K123 I, (D156H, +160N). White: Mutations (N28T,K32Q), K65N, (E96L, K97S), (P108G, D109N), (D125Y, E127S), R145E.

[0070]FIG. 25 shows circular dichroism (CD) spectra of Bet v 1.2801(wild type) and Bet v 1 (2744), recorded at nearly equal concentrations.

[0071]FIG. 26 shows histamine release in human basophil cells of Bet v1.2801 (wild type), and mutant Bet v 1 (2744).

[0072] FIGS. 27A-D shows histamine release in human basophil cells ofBet v 1.2801 (wild type), and mutant Bet v 1 (2744). (A) Donor MJ (B)Donor MH (C) Donor CJB (D) Donor MCDS.

[0073]FIG. 28 shows point mutations at the surface of Bet v 1 (2733).Grey: Back bone+Amino acid residues 95-100% conserved among Fagales.Black: Point mutations: Y5V, N28T, K32Q, E45S, K65N, N78K, K97S, K103V,P108G, K134E, R145E, D156H, +160N

[0074]FIG. 29 shows the proliferation of patient Peripheral BloodLymphocytes expressed as Stimulation Index (SI) for various Bet v 1preparations.

[0075]FIG. 30 shows the cytokine profile of T cells stimulated withvarious Bet v 1 preparations from a patient with a Th0 profile.

[0076]FIG. 31 shows the cytokine profile of T cells stimulated withvarious Bet v 1 preparations from a patient with a Th1 profile.

[0077]FIG. 32 shows the cytokine profile of T cells stimulated withvarious Bet v 1 preparations from a patient with a Th2 profile.

[0078]FIG. 33 shows Circular dichroism (CD) spectroscopy of rBet v1.2801() (wildtype) and the rBet v 1 3007) mutant [Δ] with 12mutations, recorded at equal concentrations. Overlay of circulardichroism (CD) spectra obtained at 15° C. are shown.

[0079]FIG. 34 shows the inhibition of the binding of biotinylated rBet v1.2801 to pooled IgE serum from birch allergic patients by rBet v 1.2801() (wildtype) or mutated rBet v 1 (3007) [Δ] with 12 mutations.

OBJECT OF THE INVENTION

[0080] The object of the invention is to provide improved recombinantmutant allergen proteins.

[0081] Rationale Behind the Present Invention

[0082] The current invention is based on a unique rationale. Accordingto this rationale the mechanism of successful allergy vaccination is notan alteration of the ongoing Th2-type immune response, but rather aparallel initiation of a new immune response involving tertiary epitoperecognition by B-cells and antibody formation. It is believed that thisnew immune response is partly a Th1-type immune response. When thevaccine (or pharmaceutical compositions) is administered through anotherroute than the airways, it is hypothesised, that the new immune responseevolves in a location physically separated from the ongoing Th2 responsethereby enabling the two responses to exist in parallel.

[0083] Furthermore, the invention is based on the finding that allergicsymptoms are triggered by the cross-linking of allergen with at leasttwo specific IgE's bound to the surface of effector cells, i.e. mastcells and basophils, via the high affinity IgE receptor, FcεRI. Forillustration, we refer to FIG. 1, which depicts a theoretical model ofan allergen with three IgE binding epitopes. Induction of mediatorrelease from the mast cell and hence allergic symptoms is effected byallergen-mediated cross-linking of IgE bound to the surface of the mastcell, cf. FIG. 1A. In the situation shown in FIG. 1B two of the epitopeshave been mutated so as to reduce their IgE binding ability, and hencethe allergen-mediated cross-linking is prevented. In this connection itshould be noted that allergens usually comprise more than three B cellepitopes.

[0084] In order for a mutant allergen to be able to raise the new immuneresponse, including an IgG response, the mutant allergen must compriseat least one intact epitope or an epitope, which has been altered onlymoderately. The surface topography of a moderately altered epitopepreferably resembles the original epitope, allowing new more numerousIgG antibodies to be raised. These new IgG antibodies have specificitieswhich can compete and to some degree oust IgE binding to the naturaloccurring allergen. Further, it may be assumed that the more epitopes,which have been mutated so as to eliminate or reduce their IgE bindingability, the lower the risk of allergen-mediated cross-linking andresulting allergic symptoms upon administration of an allergen vaccine.

[0085] According to this rationale it is essential that the mutantallergen has an α- carbon backbone tertiary structure which isessentially the same as that of the natural allergen.

[0086] It has previously been assumed that positions suitable formutation are located exclusively in areas consisting of conserved aminoacid residues believed to harbour dominant IgE binding epitope. However,according to the present invention it appears that surface exposed aminoacid residues suitable for mutation comprise both highly conservedresidues and residues that are not conserved or only conserved to asmaller degree. Such amino acid residues appear to be distributed overthe entire molecular surface with a tendency to form small groupingscovering a defined area on the molecular surface.

[0087] Thus, according to the present invention, surface exposed aminoacids suitable for mutation can be divided into groups as illustrated inFIG. 2. The groupings rely on the tendency of these amino acid residuesto form separate areas and these groupings are furthermore independentof the degree of conservation of the amino acid residues. Each grouprepresents a number of surface exposed amino acid residues that arefound within a limited area on the surface of the allergen. Eachindividual group most likely comprises part of at least one epitope orat least one intact epitope. Each separate group may comprise as wellamino acids positions that will result in a moderately altered epitopeupon mutation as well as amino acid positions that will result in a moredrastic alteration of the epitope upon mutation. A single amino acidresidue typically results in a moderate alteration of an epitope if theoriginal amino acid residue is substituted with an amino acid thatposseses similar chemical features (E.g. exchanging a hydrophobic aminoacid with another hydrophobic amino acid residue). In conclusion, byselecting mutations among amino acid residues from at least four of thedefined groups provides a tool for rendering it very likely that amutant allergen according to the present invention is mutated in severalB-cell epitopes and has a α-carbon backbone structure that is similar tothe naturally occurring allergen.

[0088] It is furthermore an important aspect of the present inventionthat the mutated allergen retains a continous surface region with anarea of about 400-800 Å² that contains either no mutations or onlymoderate mutations. It is believed that an allergen comprises a numberof potential binding regions for specific IgE's, wherein each region hasan area of approximately 800 Å².

[0089] The inventive idea of the present invention is based on therecognition that a mutated allergen having IgE binding reducingmutations in at least 4 defined groups, each group comprising surfaceexposed amino acids suitable for mutation, but retaining at least oneintact or moderately altered epitope, would on the one hand reduce theallergen-mediated cross-linking and on the other hand allow the raisingof an IgG response with a binding ability competitive with that of IgE.Thus, the said mutated allergen constitutes a highly advantageousallergen in that the risk of anaphylactic reactions is being stronglyreduced. The mutant allergen has the potential to be administered inrelatively higher doses improving its efficacy in generating aprotective immune response without compromising safety.

[0090] Also, the present invention is based on the recognition that avaccine comprising a mixture of different such mutated allergens,wherein ideally many or all epitopes are represented as intact epitopesor epitopes that are only moderately altered on different mutatedallergens, would be equally efficient in its ability to induceprotection against allergic symptoms as the natural occurring allergenfrom which the mutated allergens are derived.

SUMMARY OF THE INVENTION

[0091] The present invention relates to the introduction of amino acidsubstitutions into allergens. The amino acid substitutions are chosenfrom at least four groups of amino acids suitable for amino acidsubstitution. The object being to reduce the specific IgE bindingcapability of each mutated epitope while retaining at least one intactor only moderately altered epitope on the mutated allergen.

[0092] In particular the present invention relates to a recombinant Betv 1 allergen, characterised in that it is a mutant of a naturallyoccurring Bet v 1 allergen where:

[0093] a. the mutant retains essentially the same α-carbon backbonestructure as the naturally occurring allergen,

[0094] b. the mutant comprises at least four primary mutations, whicheach reduce the specific IgE binding capability of the mutated allergenas compared to the IgE binding capability of the naturally occurring Betv 1 allergen,

[0095] c. each primary mutation is a substitution of one surface-exposedamino acid residue with another residue,

[0096] d. the mutations are placed in such a manner that at least onearea of 400-800 Å² comprises either no mutations or one or more moderatemutations,

[0097] e. the primary mutations are selected from at least 4 of thefollowing 10 groups, each group comprising surface exposed amino acidpositions suitable for amino acid substitution:

[0098] group 1: A130, E131, K134, A135, K137, E138, E141, T142, R145;

[0099] group 2: V2, F3, N4, Y5, E6, T7, K 119;

[0100] group 3: D27, S39, S40, Y41, E42, N43, 144, E45, G46, N47, P50,G51, K55, D72, E73;

[0101] group 4: E8, T10, V12, P14, V105, A106, T107, P108, D109, G110,I113, K115;

[0102] group 5: A16, K20, S149, Y150, L152, A153, H154, S155, D156,Y158, N159, +160, wherein +160 represents addition of an N-terminalamino acid;

[0103] group 6: L24, D25, N28, K32;

[0104] group 7: H76, T77, N78, F79, K80, E101, K103;

[0105] group 8: K68, R70, I86, E87, E96, K97;

[0106] group 9: G1, G92, D93, T94, K123, G124, D125, H126, E127, K129;

[0107] group 10: P35, Q36, E60, G61, P63, F64, K65, Y66;

[0108] with the proviso that the recombinant Bet v 1 allergen is not oneof the following specific mutants: (Asn28Thr, Lys32Gln, Asn78Lys,Lys103Val, Arg145Glu, Asp156His, +160Asn); (Tyr5Val, Glu42Ser, Glu45Ser,Asn78Lys, Lys103Val, Lys123lle, Lys134Glu, Asp156His); (Tyr5Val,Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu); (Ala16Pro, Asn28Thr, Lys32Gln,Lys103Thr, Pro108Gly, Leu152Lys, Ala153Gly, Ser155Pro); (N28T, K32Q,N78K, K103V, P108G, R145E, D156H, +160N); (Tyr5Val, Lys134Glu, Asn28Thr,Lys32Gln, Glu45Ser, Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly,Arg145Glu, Asp156His, +160Asn); (Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser,Asn78Lys, Lys103Val, Lys231lle, Asp156His, +160Asn); (Asn28Thr,Lys32Gln, Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr,Glu127Ser, Arg145Glu); (Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G,K123I, K134E, D156H, +160N); (Y5V, E42S, E45S, K65N, N78K, K97S, K103V,K123I, K134E, D156H, +160N); and (Y5V, N28T, K32Q, E42S, E45S, K65N,N78K, K97S, K103V, P108G, K1231, K134E, D156H, +160N).

[0109] More specifically, the present invention relates to a recombinantBet v 1 allergen where the primary mutations are selected from at least4 of the following 10 groups, each group comprising surface exposedamino acid positions suitable for the following amino acidsubstitutions:

[0110] group 1: A130: A130V, A130G, A130I, A130L, A130S, A130H, A130T;E131: E131D, E131H, E131K, E131R, E131S; K134: K134R, K134H, K134S,K134Q, K1341, K134E; A135: A135V, A135G, A135I, A135L, A135S, A135H,A135T; K137: K137R, K137H, K137S, K137Q, K1371, K137E; E138: E138D,E138H, E138K, E138R, E138S, E138N; E141: E141D, E141H, E141K, E141R,E141S; T142: T142A, T142S, T142L, T142V, T142D, T142K, T142N; R145:R145K, R145H, R145T, R145D, R145E;

[0111] group 2: V2: V2A, V21, V2K, V2L, V2R, V2T; F3: F3H, F3W, F3S, F3DN4: N4H, N4K, N4M, N4Q, N4R; Y5: Y5D, Y5G, Y5H, Y51, Y5K, Y5V; E6: E6D,E6H, E6K, E6R, E6S; T7: T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119: K119R,K119H, K119S, K119Q, K1191, K119E, K119N;

[0112] group 3: D27: D27E, D27H, D27K, D27R, D27S; S39: S39T, S39L,S39V, S39D, S39K; S40: S40T, S40L, S40V, S40D, S40K; Y41: Y41D, Y41G,Y41H, Y41I, Y41K, Y41V; E42: E42S, E42D, E42H, E42K, E42R; N43: N43H,N43K, N43M, N43Q, N43R; I44: I44L, I44K, I44R, I44D; E45: E45S, E45D,E45H, E45K, E45R; G46: G46N, G46H, G46K, G46M, G46Q, G46R; N47: N47H,N47K, N47M, N47Q, N47R; P50: P50G; G51: G51N, G51H, G51K, G51M, G51Q,G51R; K55: K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72: D72E, D72S,D72H, D72R, D72K; E73: E73D, E73S, E73H, E73R, E73K;

[0113] group 4: E8: E8D, E8H, E8K, E8R, E8S; T10: T10P, T10S, T10L,T10V, T10D, T10K, T10N; V12: V12A, V12I, V12K, V12L, V12R, V12T; P14:P14G; V105: V105A, V105I, V105K, V105L, V105R, V105T; A106: A106V,A106G, A106I, A106L, A106S, A106H, A106T; T107: T107A, T107S, T107L,T107V, T107D, T107K, T107N; P108: P108G; D109: D109N D109E, D109S,D109H, D109R, D109K; G110: G110N, G110H, G110K, G110M, G110Q, G110R;I113: I113L, I113K, I113R, I113D, K115: K115R, K115H, K115S, K115Q,K151, K115E, K115N;

[0114] group 5: A16: A16V, A16G, A16I, A16L, A16S, A16H, A16T; K20:K20R, K20H, K20S, K20Q, K20I, K20E, K20N; S149: S149T, S149L, S149V,S149D, S149K; Y150: Y150T, Y150L, Y150V, Y150D, Y150K; L152: L152A,L152V, L152G, L1521, L152S, L152H, L152T; A153: A153V, A153G, A153I,A153L, A153S, A153H, A153T; H154: H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; D156: D156H, D156E, D156S, D156R,D156K; Y158: Y158D, Y158G, Y158H, Y158I, Y158K, Y158V; N159: N159H,N159K, N159M, N159Q, N159R, N159G, +160N;

[0115] group 6: L24: L24A, L24V, L24G, L24I, L24S, L24H, L24T; D25:D25E, D25H, D25K, D25R, D25S; N28: N28H, N28K, N28M, N28Q, N28R, N28T;K32: K32Q, K32R, K32N, K32H, K32S, K321, K32E;

[0116] group 7: H76: H76W, H76F, H76S, H76D; T77: T77A, T77S, T77L,T77V, T77D, T77K, T77N; N78: N78H, N78K, N78M, N78Q, N78R; F79: F79H,F79W, F79S, F79D; K80: K80R, K80H, K80S, K80Q, K801, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103: K103R, K103H, K103S, K103Q,K103I, K103E, K103V;

[0117] group 8: K68: K68R, K68H, K68S, K68Q, K681, K68E, K68N; R70:R70K, R70H, R70T, R70D, R70E, R70N; 186: 186L, I86K, I86R, I86D; E87:E87D, E87H, E87K, E87R, E87S, E87A; E96: E96D, E96H, E96K, E96R, E96S,E96L; K97: K97R, K97H, K97S, K97Q, K971, K97E;

[0118] group 9: G1: G1N, G1H, G1K, G1M, G1Q, G1R; G92: G92N, G92H, G92K,G92M, G92Q, G92R; D93: D93N, D93E, D93S, D93H, D93R, D93K; T94: T94A,T94S, T94L, T94V, T94D, T94K, T94N; K123: K123R, K123H, K123S, K123Q,K123I, K123E; G124: G124N, G124H, G124K, G124M, G124Q, G124R; D125:D125E, D125H, D125K, D125R, D125S, D125Y; H126: H126W, H126F, H126S,H126D; E127: E127D, E127H, E127K, E127R, E127S; K129: K129R, K129H,K129S, K129Q, K129I, K129E, K129N;

[0119] group 10: P35: P35G; Q36: Q36K, Q36R, Q36N, Q36H, Q36S, Q36I,Q36E; E60: E60H, E60K, E60M, E60Q, E60R; G61: G61N, G61H, G61K, G61M,G61Q, G61R; P63: P63G; F64: F64H, F64W, F64S, F64D; K65: K65R, K65H,K65S, K65Q, K651, K65E, K65N; Y66: Y66D, Y66G, Y66H, Y661, Y66K, Y66V.

[0120] The present invention further relates to a recombinant Bet v 1mutant allergen comprising substitutions that are selected from at leastfour of the following 10 groups:

[0121] Group 1: A130V, K134E, E141N,

[0122] Group 2: V2L, Y5V, E6S, K119N,

[0123] Group 3: E42S, E45S, N47K, K55N, E73S, E73T, E73S,

[0124] Group 4: E8S, T10P, P14G, P108G, D109N, K115N,

[0125] Group 5: A16G, K20S, S149T L152A A153V, S155T, N159G, +160N,

[0126] Group 6: L24A, D25E, N28T, K32Q,

[0127] Group 7: T77A, T77N, N78K, K103V,

[0128] Group 8: R70N, E87A, E96S, K97S,

[0129] Group 9: D93S, K1231, D125Y, K129N,

[0130] Group 10: Q36N, E60S, G61S, P63G.

[0131] The present invention further relates to a recombinant Bet v 1mutant allergen comprising substitutions that are selected from at leastfour of the following 10 groups:

[0132] Group 1: K134E,

[0133] Group 2: Y5V, K119N, V2L,

[0134] Group 3: E45S, E42S, K55N, N47K, E73S,

[0135] Group 4: E96S, K97S, P108G, D109N, T10N, K115N, P14G,

[0136] Group 5: N159G, +160N, S149T, A153V, L152A, A16G, K20S,

[0137] Group 6: N28T, K32Q, L24A,

[0138] Group 7: K103V, T77N, N78K,

[0139] Group 8: E96S, K97S, E87A,

[0140] Group 9: K129N, D125Y, K123I, D93S,

[0141] Group 10: E60S, Q36N, G61S, P63G.

[0142] The present invention further relates to the following:

[0143] Recombinant Bet v 1 allergens variants that can be used as apharmaceutical and for preparing a pharmaceutical for preventing and/ortreating birch pollen allergy.

[0144] A composition comprising two or more different recombinant mutantBet v 1 allergen variants according to the present invention whereineach variant has at least one primary mutation, which is absent in atleast one of the other variants. The composition comprises 2-12,preferably 3-10, more preferably 4-9 and most preferably 5-8 variants. Acomposition according to the present invention can be used as apharmaceutical and for preparing a pharmaceutical for preventing and/ortreating birch pollen allergy. The pharmaceutical composition preferablycomprises a pharmaceutically acceptable carrier, and/or excipient, andoptionally an adjuvant.

[0145] A pharmaceutical composition in the form of a vaccine againstallergic reactions elicited by a naturally occurring Bet v 1 allergen inpatients suffering from birch pollen allergy.

[0146] Methods of generating an immune response in a subject comprisingadministering to a subject a recombinant allergen, a composition, or apharmaceutical composition.

[0147] Vaccination or treatment of a subject comprises administering tothe subject a recombinant allergen, a composition, or a pharmaceuticalcomposition.

[0148] A method for preparing a pharmaceutical composition comprisingmixing a recombinant allergen, or a composition with pharmaceuticallyacceptable substances, and/or excipients.

[0149] A method for the treatment, prevention or alleviation of allergicreactions in a subject that comprises administering to a subject arecombinant Bet v 1 allergen, a composition, or a pharmaceuticalcomposition.

[0150] A method of preparing a recombinant Bet v 1 allergencharacterised in that the substitution of amino acids is carried out bysite-directed mutagenesis, or DNA shuffling (molecular breeding)(Punnonen et al., ref. 25).

[0151] A DNA sequence encoding a recombinant Bet v 1 allergen, aderivative thereof, a partial sequence thereof, a degenerated sequencethereof or a sequence which hybridises thereto under stringentconditions, wherein said derivative, partial sequence, degeneratedsequence or hybridising sequence encodes a peptide having at least one Bcell epitope.

[0152] A DNA sequence which is a derivative of the DNA sequence encodingthe naturally occurring allergen. The DNA sequence encoding thederivative is obtained by site-directed mutagenesis of the DNA encodingthe naturally occurring Bet v 1 allergen.

[0153] An expression vector comprising DNA encoding a recombinant Bet v1 variant, a host cell comprising the expression vector, and a method ofproducing a recombinant mutant Bet v 1 allergen comprising cultivatingthe host cell.

[0154] A recombinant Bet v 1 allergen or a recombinant Bet v 1 allergenthat is encoded by the DNA sequence comprises at least one T celleepitope capable of stimulating a T cell clone or T cell line specificfor the naturally occurring Bet v 1 allergen.

[0155] A diagnostic assay for assessing relevance, safety, or outcome oftherapy of a subject using a recombinant mutant Bet v 1 allergen or acomposition, wherein an IgE containing sample of a subject is mixed withsaid mutant or said composition and assessed for the level of reactivitybetween the IgE in said sample and said mutant.

DETAILED DESCRIPTION OF THE INVENTION

[0156] In connection with the present invention the expression “reducethe specific IgE binding capability as compared to the IgE bindingcapability of the naturally occurring allergen” means that the reductionis measurable in a statistically significant manner (p<0.05) in at leastone immunoassay using serum from a subject allergic to thenatural-occurring allergen. Preferably, the IgE binding capability isreduced by at least 10%, more preferably at least 30%, more preferablyat least 50%, and most preferably at least 70%.

[0157] The expression “surface-exposed amino acid” means that the aminoacid residue is located at the surface of the three-dimensionalstructure in such a manner that when the allergen is in solution atleast a part of at least one atom of the amino acid residue isaccessible for contact with the surrounding solvent. Preferably, theamino acid residue in the three-dimensional structure has a solvent(water) accessibility of at least 20%, suitably at least 30%, moresuitably at least 40% and most preferably at least 50%.

[0158] The expression “solvent accessibility” is defined as the area ofthe molecule accessible to a sphere with a radius comparable to asolvent (water, r=1.4 Å) molecule. The expressions “surface-exposed” and“solvent-exposed” are used interchangeably.

[0159] “Group of amino acids” should be understood as division ofsurface exposed amino acids suitable for mutation into groups. Eachgroup represents a number of surface exposed amino acid residues thatare found within a limited area on the surface of the allergen. Anindividual group comprises a number of amino acids that are part of atleast one epitope. An individual group may also cover an area thatcomprises an entire epitope. One or more mutations within a single groupis defined as one primary mutation. A mutated allergen with at leastfour primary mutations thus ensures that several epitopes will have alowered IgE binding affinity. Mutation of amino acids from at least fourgroups may furthermore ensure an approximately even distribution ofmutations on the molecular surface and ensure that several epitopes aremutated and thus resulting in a lowered IgE binding affinity of severalepitopes compared to mutants with less than four mutations.

[0160] The expression “the taxonomic species from which said naturallyoccurring allergen originates” means species within the taxonomic genus,preferably the subfamily, more preferably the family, more preferablythe superfamily, more preferably the legion, more preferably thesuborder and most preferably the order from which said naturallyoccurring allergen originates.

[0161] The expression “moderately altered epitopes” means epitopes thatretain essentially the same tertiary structure and surface topography asthe corresponding unmutated epitopes. Moderate alterations are,generally speaking, achieved by exchanging an amino acid with anotheramino acid with similar chemical characteristics as the original aminoacid. One way of achieving this is by exchanging one or more surfaceexposed amino acids with amino acids that might be found within thetaxonomic order wherein the naturally occurring allergen is found. Amoderately altered epitope might also contain amino acid substitutionswhere one or more of the substituted amino acid is not found within thetaxonomic order wherein the naturally occurring allergen is found, aslong as the substitution only slightly affects the tertiary structure ofthe epitope and/or the IgE binding affinity. The mutated allergen can beevaluated with respect to e.g. structure and IgE binding affinitysubsequently. As opposed to the moderately altered epitopes are epitopesthat are altered in a more drastic manner, e.g. mutations thatsignificantly reduce the IgE binding affinity. Typically, drasticalterations of epitopes comprise amino acid substitutions where one ormore amino acids have been exchanged with amio acids with differentchemical properties.

[0162] Furthermore, the expression “the mutant allergen havingessentially the same α-carbon backbone tertiary structure as thenaturally occurring allergen” means that when comparing the structuresof the mutant and the naturally occurring allergen, the average rootmean square deviation of the atomic coordinates is preferably below 2 Å.Conservation of α-carbon backbone tertiary structure is best determinedby obtaining identical structures by x-ray crystallography or NMR beforeand after mutagenesis. In absence of structural data describing themutant indistinguishable CD-spectra or immunochemical data, e.g.antibody reactivity, may render conservation of α-carbon backbonetertiary structure probable, if compared to the data obtained byanalysis of a structurally determined molecule.

[0163] In connection with the present invention the expression“mutation” means the deletion, substitution or addition of an amino acidin comparison to the amino acid sequence of the naturally occurringallergen. The terms “mutation” and “substitution” are usedinterchangeably. A recombinant mutated Bet v 1 allergen according to theinvention may furthermore comprise amino acid insertions or amino aciddeletion in particular surface exposed regions of the molecules e.g.“loop regions”. Loop regions connect secondary structure elements e.g.β-sheet, α-helixes and random coil structures. Loop regions in Bet v 1are: Val12 to ala16, val33 to ser40, glu45 to Thr52, pro54 to tyr66,his76 to asn78, gly89 to glu96, val105 to gly111, thr122 to glu131.Mutant variants may comprise 1-5, more preferable 1-3 most preferably1-2 substitutions in a loop region.

[0164] A primary mutation is defined as one or more mutations within asingle group of surface exposed amino acids suitable for substitution.Each group of at least one mutated amino acids will have reduced IgEbinding affinity as compared to the same group without mutations.Preferably, the recombinant allergen according to the inventioncomprises from 5 to 10, preferably from 6 to 10, more preferably from 7to 10, and most preferably from 8 to 10 primary mutations.

[0165] Secondary mutations are defined as additional mutations within asingle group. The recombinant allergen preferably comprises a number ofsecondary mutations, which each reduce the specific IgE bindingcapability of the mutated allergen as compared to the binding capabilityof the said naturally occurring allergen. Thus, a primary mutation thatcomprises several secondary mutations will in many cases have a morereduced IgE binding affinity than a primary mutation that has only onemutation. The recombinant allergen according to the invention comprisesfrom 1 to 15, preferably 1-10 and most preferably 1-5 secondarymutations per primary mutation.

[0166] Conserved residues: Conserved residues in the naturally occurringallergen are conserved with more than 70%, preferably 80% and mostpreferably 90% identity in all known homologous proteins within thespecies from which said allergen originates. Amino acid residues thatare highly solvent exposed and conserved constitute targets forsubstitution.

[0167] Another way of assessing the reduced IgE binding and the reducedability of mediating cross-linking of the mutant are the capability ofthe mutant to initiate Histamine Release (HR). The release of Histaminecan be measured in several Histamine releasing assays. The reducedHistamine release of the mutants originates from reduced affinity towardthe specific IgE bound to the cell surface as well as their reducedability to facilitate cross-linking. HR is preferably reduced by 5-100%,more preferably 25-100%, more preferably 50-100% and most preferably75-100% for the mutants of the invention in comparison to the naturallyoccurring allergens.

[0168] In a preferred embodiment of the invention, a surface regioncomprising no mutation or only moderate mutations has an area of 800 Å²,preferably 600 Å², more preferably 500 Å² and most preferably 400 Å².Typically, a surface region with an area of 800 Å² comprising nomutation or only moderate mutations comprises atoms of 15-25 amino acidresidues.

[0169] In another embodiment, at least one of the amino acid residues tobe incorporated into the mutant allergen does not occur in the sameposition in the amino acid sequence of any known homologous proteinwithin the taxonomic genus, preferably the subfamily, more preferablythe family, more preferably the superfamily, more preferably the legion,more preferably the suborder and most preferably the order from whichsaid naturally occurring allergen originates.

[0170] According to the invention, the surface-exposed amino acidresidues are ranked with respect to solvent accessibility, and at leastfour amino acids among the more solvent accessible ones are substituted.

[0171] In a further embodiment, a recombinant allergen is characterisedin that the surface-exposed amino acid residues are ranked with respectto degree of conversation in all known homologous proteins within thespecies from which said naturally occurring allergen originates, andthat one or more surface exposed amino acids among the more conservedones are substituted.

[0172] The principle disclosed in the present invention comprisesmutation of surface exposed amino acid residues selected from at leastfour groups of amino acids, wherein each group represents separate areason the surface on the molecule. This principle may also be applied toallergens other than Bet v 1. A recombinant allergen according to theinvention may suitably be a mutant of an inhalation allergen originatingi.a. from trees, grasses, herbs, fungi, house dust mites, cockroachesand animal hair and dandruff. Important pollen allergens from trees,grasses and herbs are such originating from the taxonomic orders ofFagales, Oleales and Pinales including i.a. birch (Betula), alder(Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), theorder of Poales including i.a. grasses of the genera Lolium, Phleum,Poa, Cynodon, Dactylis and Secale, the orders of Asterales and Urticalesincluding i.a. herbs of the genera Ambrosia and Artemisia. Importantinhalation allergens from fungi are i.a. such originating from thegenera Alternaria and Cladosporium. Other important inhalation allergensare those from house dust mites of the genus Dermatophagoides, thosefrom cockroaches and those from mammals such as cat, dog and horse.Further, recombinant allergens according to the invention may be mutantsof venom allergens including such originating from stinging or bitinginsects such as those from the taxonomic order of Hymenoptera includingbees (superfamily Apidae), wasps (superfamily Vespidea), and ants(superfamily Formicoidae).

[0173] Specific allergen components include e.g. Bet v 1 (B. verrucosa,birch), Aln g 1 (Alnus glutinosa, alder), Cor a 1 (Corylus avelana,hazel) and Car b 1 (Carpinus betulus, hornbeam) of the Fagales order.Others are Cry j 1 (Pinales), Amb a 1 and 2, Art v 1 (Asterales), Par j1 (Urticales), Ole e 1 (Oleales), Ave e 1, Cyn d 1, Dac g 1, Fes p 1,Holl 1, Lol p 1 and 5, Pas n 1, Phl p 1 and 5, Poa p 1, 2 and 5, Sec c 1and 5, and Sor h 1 (various grass pollens), Alt a 1 and Cla h 1 (fungi),Der f 1 and 2, Derp 1 and 2 (house dust mites, D. farinae and D.pteronyssinus, respectively), Eur m 1 (mite, Euroglyphus maynel), (Lep d1 and 2 (Lepidoglyphus destructor; storage mite), Bla g 1 and 2, Per a 1(cockroaches, Blatella germanica and Periplaneta americana,respectively), Fel d 1 (cat), Can f 1 (dog), Equ c 1, 2 and 3 (horse),Apis m 1 and 2 (honeybee), Ves v 1, 2 and 5, Pol a 1, 2 and 5 (allwasps) and Sol i 1, 2, 3 and 4 (fire ant).

[0174] Some Examples of Adding Further Substitutions to a Given Mutant

[0175] In one embodiment of the invention further substitutions areadded to mutant allergens in such a way that it is ensured that thesubstitutions of the final mutant allergen are essentially evenlydistributed on the molecular surface and that the groups containessentially the same number of introduced mutations. This is illustratedin the following examples where mutants comprising specificsubstitutions preferably should have added further substitutions from alist where the succession of amino acids reflects the preferred order ofadding more substitutions. Without limiting the present invention, theseexamples represent one application of how to design mutants and the manskilled in the art might very well choose a somewhat different approachin order to ensure an even distribution of substitutions. Mutants maythus be designed comprising one or more substitutions from the listsgiven below.

[0176] Bet v 1 mutant (“3004A”) allergens comprising the followingsubstitutions: Y5V, E45S, N78K, K97S, K103V, K134E, +160N. Furthersubstitutions may comprise one or more of the following: E8 or K115,D125 or H126, E138 or K137 or E141, D25 or N28, E87 or K55, S155 or H154or N159, N47 or P50 or H76 or N43 or I44 or R70, E73 or P50 or D72,A130, N28 or D25, P108, V2 or K119 or N4 or E6 or E96.

[0177] Bet v 1 mutant (“3004B”) allergens comprising the followingsubstitutions: Y5V, E45S, L62F, N78K, K97S, K103V, K134E, +160N. Furthersubstitutions may comprise one or more of the following: T10P, K65N, N28or D25 or K32Q or E141X or K137X or E138X, D125X or K1231 or H126, P108Xor D109N, E42S or K55X or I44X or N43X, E73X or D72X, E87X, E96X orK119, A130X, V2X or E6X, E8X or K115, N47X or P50X or R70X or H76X orT77A, S155X or D156H or N159X, E6X or V2X.

[0178] Bet v 1 allergen mutants (“3005A”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, K134E,+160N. Further substitutions may comprise one or more of the following:E8X or K115X, D125 or H126, E138X or K137X or E141X, E87X or K55X,S155Xor H154X or N159X, N47X or P50X or H76X or N43X or I44X or R70X,E73X or P50X or D72X, A130X, D25X, P108X, V2X or K119X or N4X or E6X orE96X.

[0179] Bet v 1 allergen mutants (“3005B”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, K134E,+160N. Further substitutions may comprise one or more of the following:T10P, K65N, E141X or K137X or E138X, D125X or K123I or H126X, P108X orD109N, E42S or K55X or I44X or N43X, E73X or D72X, E87X, E96X or K119X,A130X, V2X or E6X, E8X or K115X, N47X or P50X or R70X or H76X or T77A,S155X or D156H or N159X, E6X or V2X.

[0180] Bet v 1 allergen mutants (“3006A”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, N78K, E87S, K97S, K103V, K134E,N159G, +160N. Further substitutions may comprise one or more of thefollowing: K55, A138 or K137 or E141, D125 or H126, P108, V2 or N4 orK119 or E6, S155 or H154, N47 or P50 or H76, E73, R70, A130, E8 or K115,E96.

[0181] Bet v 1 allergen mutants (“3006B”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, N78K, E87S, K97S, K103V, K134E,N159G, +160N. Further substitutions may comprise one or more of thefollowing: K65N, T10P, D125, K123I, P108, D109N, N47 or P50 or H76, E138or K137 or E141, E42S or K55 or I44 or N43, S155 or D156H, E73 or D72,E6 or V2, E96.

[0182] Bet v 1 allergen mutants (“3007A”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, P108G,D125Y, K134E, +160N. Further substitutions may comprise one or more ofthe following: E87, E141, E138, K55, N47 or N43X or 144 or H76, S155 orH154, A130, E8, E73, V2 or K119, D25.

[0183] Bet v 1 allergen mutants (“3007B”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, N78K, K97S, K103V, P108G,D125Y, K134E, +160N. Further substitutions may comprise one or more ofthe following: K65N, T10P or E8, E87, S155 or D156H, E138, E141, E42S,A130, E8 or T10P, N47, H76, R70, E96.

[0184] Bet v 1 allergen mutants (“3008A”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, E73S, E96S, P108G, D125Y,N159G, +160N. Further substitutions may comprise one or more of thefollowing: E134, N78, E87, K119, E8, K55, E138, E141, S155, N47, E6,K103, D25, A130, V2, R70.

[0185] Bet v 1 allergen mutants (“3008B”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, E73S, E96S, P108G, D125Y,N159G, +160N. Further substitutions may comprise one or more of thefollowing: K65N or K55, T10P or E8 or E141, E138 or K134, E87, E42S orK55 or I44, S155 or D156H, N78, K119 or V2 or N4, N47 or P50, H76 orT77A, A130, D25, E6 or K115 or K103.

[0186] Bet v 1 allergen mutants (“3009A”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, E96S, P108G, +160N. Furthersubstitutions may comprise one or more of the following: E134, N78, E87,K1 19, E8, K55, E138, E141, S155, N47, E6, K103, D25, A130, V2, R70.

[0187] Bet v 1 allergen mutants (“3009B”) comprising the followingsubstitutions: Y5V, N28T, K32Q, E45S, L62F, E96S, P108G, +160N. Furthersubstitutions may comprise one or more of the following: N78 or T77A,K103, E134 or E138, K65N or K55, T10P, D125 or H126, S155 or D156H orHIS154, K119 or V2, E87, N47 or P50 or H76, E42S or K55, I44 or N43,A130.

[0188] Loop Mutations:

[0189] In another embodiment of the invention mutant allergens accordingto the invention furthermore comprise amino acid insertions or aminoacid deletion in particular surface exposed regions of the moleculese.g. loop regions. Loop regions connect secondary structure elementse.g. β-sheet, α-helixes and random coil structures. Loop regions in Betv 1 are: val12 to ala16, val33 to ser40, glu45 to Thr52, pro54 to tyr66,his76 to asn78, gly89 to glu96, val105 to gly111, thr122 to glu131.Mutant variants according to this embodiment comprise 1-5, morepreferable 1-3 most preferably 1-2 substitutions in a loop region. In apreferred embodiment, mutant allergens comprise at least four mutationsselected from the 10 groups as well as a number of additional“loop-mutations”. Examples of such “loop mutations” , wherein xrepresents an added amino acid residue, are:

[0190] Bet v 1 (3007-L1) with an amino acid insertion between residueE60 and residue G61 (SEQ ID NO 1):GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTIKKISFPExGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVATGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVES YLLAHSDAYNN

[0191] Bet v 1 (3007-L2) with amino acid insertion between residue D93and residue T94 (SEQ ID NO 2):GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDxTLESISNEIVIVATGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVES YLLAHSDAYNN

[0192] Bet v 1 (3007-L3) with amino acid insertion between residue V12and residue I13 (SEQ ID NO 3):GVFNVETETTSVxIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVATGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVES YLLAHSDAYNN

[0193] Bet v 1 (3007-L4) with amino acid insertions between residue I56and residue S57 and between residue K65 and residue T66 (SEQ ID NO 4):GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTIKKIxSFPExGFPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLESISNEIVIVATGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVE SYLLAHSDAYNN

[0194] Bet v 1 (3007-L5) with amino acid deletion of residue G111 (SEQID NO 5): GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVATGDGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYL LAHSDAYNN

[0195] Method of Preparing a Recombinant Allergen According to theInvention

[0196] The surface-exposed amino acids suitable for substitution inaccordance with the present invention may be identified on the basis ofinformation of their solvent (water) accessibility, which expresses theextent of surface exposure. A preferred embodiment of the method of theinvention is characterised in ranking the said identified amino acidresidues with respect to solvent accessibility and substituting one ormore amino acids among the more solvent accessible ones.

[0197] Furthermore, another embodiment of the method of the invention ischaracterised in ranking the identified amino acid residues with respectto degree of conversation in all known homologous proteins within thespecies from which said naturally occurring allergen originates andsubstituting one or more amino acids among the more conserved ones.

[0198] A further preferred embodiment of the method of the inventioncomprises selecting the identified amino acids so as to form a mutantallergen, which has essentially the same α-carbon backbone tertiarystructure as said naturally occurring allergen.

[0199] Another preferred embodiment of the method of the invention ischaracterised in that the substitution of amino acid residues is carriedout by site-directed mutagenesis.

[0200] An alternative preferred embodiment of the method of theinvention is characterised in that the substitution of amino acidresidues is carried out by DNA shuffling or by setting up a librarycomprising suitable positions and their preferred substitutents.

[0201] Criteria for Substitution

[0202] For molecules for which the tertiary structure has beendetermined (e.g. by x-ray crystallography, or NMR electron microscopy),the mutant carrying the substituted amino acid(s) should preferablyfulfil the following criteria:

[0203] 1. The overall α-carbon backbone tertiary structure of therecombinant mutant is preferably conserved. Conserved is defined as anaverage root mean square deviation of the atomic coordinates below 2 Åwhen comparing the structures of the mutated allergen and the naturallyoccurring allergen. This is important for two reasons: a) It isanticipated that the entire surface of the natural allergen constitutesan overlapping continuum of potential antibody-binding epitopes. Themajority of the surface of the molecule is not affected by thesubstitution(s), and thus retain its antibody-binding inducingproperties, which is important for the generation of new protectiveantibody specificities being directed at epitopes present also on thenatural allergen. b) Stability, both concerning shelf-life and uponinjection into body fluids.

[0204] Conservation of α-carbon backbone tertiary structure is bestdetermined by obtaining identical structures by x-ray crystallography orNMR before and after mutagenesis. In absence of structural datadescribing the mutant indistinguishable CD-spectra or immunochemicaldata, e.g. antibody reactivity, may render conservation of α-carbonbackbone tertiary structure probable, if compared to the data obtainedby analysis of a structurally determined molecule.

[0205] 2. The amino acids to be substituted are preferably located atthe surface, and thus accessible for antibody-binding. Amino acidslocated on the surface in the three-dimensional structure usually have asolvent (water) accessibility of at least 20%, suitably 20-80%, moresuitably 30-80%. Solvent accessibility is defined as the area of themolecule accessible to a sphere with a radius comparable to a solvent(water, r=1.4 Å) molecule.

[0206] 3. The substituted amino acids are selected from at least fourgroups. Each group represents a number of preferred surface exposedamino acid residues that are found within a limited area on the surfaceof the allergen.

[0207] One or more mutations within a single group is defined as oneprimary mutation. An individual group comprises a number of amino acidsthat are part of at least one epitope. An individual group may alsocomprise an entire epitope. A mutated allergen with at least fourprimary mutations thus ensures that several epitopes will have a loweredIgE binding affinity. Mutation of amino acids from at least four groupsfurthermore ensures an approximately even distribution of mutations onthe molecular surface and it ensures that several epitopes will becomemutated and thus obtaining a lowered IgE binding affinity of severalepitopes.

[0208] With an object of essentially retaining the three-dimensionalstructure of the allergen, the amino acid to be incorporated may beselected on the basis of a comparison with a protein, which is astructural homologue to the allergen, e.g. a protein, which belongs tothe same taxonomic order as the allergen, and which does not have anycross-reactivity with the allergen.

[0209] Vaccines:

[0210] Preparation of vaccines is generally well known in the art.Vaccines are typically prepared as injectables either as liquidsolutions or suspensions. Such vaccine may also be emulsified orformulated so as to enable nasal administration as well as oral,including buccal and sublingual, administration. The immunogeniccomponent in question (the recombinant allergen as defined herein) maysuitably be mixed with excipients which are pharmaceutically acceptableand further compatible with the active ingredient. Examples of suitableexcipients are water, saline, dextrose, glycerol, ethanol and the likeas well as combinations thereof. The vaccine may additionally containother substances such as wetting agents, emulsifying agents, bufferingagents or adjuvants enhancing the effectiveness of the vaccine.

[0211] Vaccines are most frequently administered parenterally bysubcutaneous or intramuscular injection. Formulations which are suitablefor administration by another route include oral formulations andsuppositories. Vaccines for oral administration may suitably beformulated with excipients normally employed for such formulations, e.g.pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecomposition can be formulated as solutions, suspensions, emulsions,tablets, pills, capsules, sustained release formulations, aerosols,powders, or granulates.

[0212] The vaccines are administered in a way so as to be compatiblewith the dosage formulation and in such amount as will betherapeutically effective and immunogenic. The quantity of activecomponent contained within the vaccine depends on the subject to betreated, i.a. the capability of the subject's immune system to respondto the treatment, the route of administration and the age and weight ofthe subject. Suitable dosage ranges can vary within the range from about0.0001 μg to 1000 μg.

[0213] As mentioned above, an increased effect may be obtained by addingadjuvants to the formulation. Examples of such adjuvants are aluminumhydroxide and phosphate (alum) or calcium phosphate as a 0.05 to 0.1percent solution in phosphate buffered saline, synthetic polymers ofsugars or polylactid glycolid (PLG) used as 0.25 percent solution.Mixture with bacterial cells such as C. parvum, endotoxins orlipopolysaccharide components of gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide monoaleate(Aracel A) or emulsion with 20 percent solution of a perfluorocarbon(e.g. Fluosol-DA) used as a block substitute may also be employed. Oilemulsions, such as MF-59 may also be used. Other adjuvants such asFreund's complete and incomplete adjuvants as well as saponins, such asQuilA, Qs-21 and ISCOM, and RIBI may also be used.

[0214] Most often, multiple administrations of the vaccine will benecessary to ensure an effect. Frequently, the vaccine is administeredas an initial administration followed by subsequent inoculations orother administrations. The number of vaccinations will typically be inthe range of from 1 to 50, usually not exceeding 35 vaccinations.Vaccination will normally be performed from biweekly to bimonthly for aperiod of 3 months to 5 years. This is contemplated to give desiredlevel of prophylactic or therapeutic effect.

[0215] The recombinant allergen may be used as a pharmaceuticalpreparation, which is suitable for providing a certain protectionagainst allergic responses during the period of the year where symptomsoccur (prophylaxis). Usually, the treatment will have to be repeatedevery year to maintain the protective effect. Preparations formulatedfor nasal, oral and sublingual application are particular suited forthis purpose.

[0216] DNA According to the Invention

[0217] The DNA sequence of the invention is a mutant of a DNA sequenceencoding a naturally occurring Bet v 1 allergen. Examples of naturallyoccurring Bet v 1 molecules are SEQ ID NO 1 (data base accession numberZ80104) and SEQ ID NO 2 (data base accession number P15494). Other Bet v1 variants include Bet v 1 sequences with the following data baseaccession numbers: P15494=X15877=Z80106, Z80101, AJ002107, Z72429,AJ002108, Z80105, Z80100, Z80103, AJ001555, Z80102, AJ002110, Z72436,P43183=X77271, Z72430, AJ002106, P43178=X77267, P43179=X77268,P43177=X77266, Z72438, P43180=X77269, AJ001551, P43185=X77273, AJ001557,Z72434, AJ001556, Z72433=P43186, AJ001554, X81972, Z72431,P45431=X77200, P43184=X77272, P43176=X77265, S47250, S47251, Z72435,Z72439, Z72437, and S47249.

[0218] Preferably, the DNA derivative is obtained by site-directed orrandom or semi random mutagenesis of the DNA encoding the naturallyoccurring allergen.

[0219] A “mutant library” is a library of mutant allergens. This libraryis constructed using degenerated DNA oligonucleotide primers that allowintroduction of none, a single or several different amino acid residuesin each position. Such a library approach allows amino acid residues tobe either conservatively or non-conservatively substituted. Asstructural integrity may be less affected by conserved mutationsintroduction of such “soft” or moderate mutations in certain positionsmay increase the changes of generating stable mutants. Construction ofmutant libraries may be one way to overcome problems with proteinstability of mutated allergens caused by a single or a certaincombination of mutations. A “semi-random library” means that positionsto be mutated are confined to amino acid residues, which are surfaceexposed.

[0220] This approach further enhances the probability of obtainingstable mutant allergens. “Semi-random” can also mean that the primersdesigned allow for a selected number of amino acid residues to besubstituted in the chosen position. The two semi-random approaches canbe used independently or in combination. Theoretically, a libraryaccording to the invention comprises a number of rBet v 1 mutantallergens each having at least 4 amino acid substitutions compared tonon-mutated Bet v 1.

[0221] In one embodiment a semi-random library based on rBet v 1 (2744)(mutated in positions Y5, E42, E45, N78, K103, K123, K134, D156, +160)and rBet v1 (2628) (mutated in positions Y5, E45, K65, K97, K134) wasconstructed where an additional 7 target positions on the allergensurface were targeted: T10, K20, Q36, E73, E87, K129 and S149. Theseseven positions were selected from surface areas that are outsidecoherent surface areas that are common among Fagales allergens. Thelibrary was based on the use of degenerated DNA oligonucleotide primersallowing introduction of several different amino acid residues in eachposition. In addition, several mutated amino acid residue positions inrBet v 1 (2744) and rBet v1 (2628) could either be maintained or mutatedback to the residues found in WT rBet v 1.2801.

[0222] I another embodiment a semi-random library based on rBet v 1(2744) and rBet v1 (2628) and rBet v 1 (2595) i.e. N28, K32, E45, P108was constructed where an additional 7 target positions on the allergensurface were targeted: T10, K20, Q36, E73, E87, K129 and S149.

[0223] Mutants:

[0224] Examples of specific Bet v 1 allergen mutants according to thepresent invention are listed below. Mutated amino acid positions areindicated in bold small print: Bet v 1 (“3004”):GVFNvETETTSVIPAARLFKAFILDGDNLFPKVAPQ (SEQ ID NO 6)AISSVsNIEGNGGPGTIKKISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLL RAVESYLLAHSDAYNn Bet v 1 (“3005”):GVFNvETEITSVIPAARLFKAFILDGDtLFPqVAPQ (SEQ ID NO 7)AISSVENIsGNGGPGTIKKISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLL RAVESYLLAHSDAYNn Bet v 1 (“3007”):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQ (SEQ ID NO 8)AISSVENIsGNGGPGTIKKISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKAEQVeASKEMGETLL RAVESYLLAHSDAYNn Bet v 1 (“3009”):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQ (SEQ ID NO 9)AISSVENIsGNGGPGTIKKISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLsKISNEIKIVATgDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLL RAVESYLLAHSDAYNn Bet v 1 (“3006”):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQ (SEQ ID NO 10)AISSVENIsGNGGPGTIKKISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLL RAVESYLLAHSDAYgn Bet v 1 (“3008”):GVFNvETETTSVIPAARLFKAFILDGDtLFPkVAPQ (SEQ ID NO 11)AISSVENIsGNGGPGTIKKISFPEGfPFKYVKDRVDsVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgDGGSILKISNKYHTKGyHEVKAEQVKASKEMGETLL RAVESYLLAHSDAYgn

[0225] The present invention furthermore comprises the followingspecific mutants: Bet v 1 (“3005-7”): Y5V, N28T, K32Q, E45S, N78K, K97S,K103V, K134E, +160N, E8S, D125Y, E141S, D25T, E87A, S155T, N47K, K55N.GVFNvETsTTSVIPAARLFKAFILtGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS (SEQ ID NO 12)FPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLEsISNEIVlVATPDGGSILKISNKYHTKGyHEVKAEQVeASKEMGsTLLRAVESYLLAHtDAYNn Bet v 1 (“3005-12”):Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, K134E, +160N, E8S, D125Y,E14IS, D25T, E87A, S155T, N47K, K55N, E73T, A13OV, P108G, V2L.GIFNvETsTTSVIPAARLFKAFILtGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS (SEQ ID NO 13)FPEGLPFKYVKDRVDtVDHTkFkYNYSVIaGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKvEQVeASKEMGsTLLRAVESYLLAHtDAYNn Bet v 1 (“3005-22”):Y5V, N28T, K320, E45S, N78K, K97S, K103V, K134E, +160N, T10K, K65N,E141N, K1231, D1O9N, E42S, EJ3T, E87A, V2L, N47K.GIFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKI (SEQ ID NO 14)SFPEGLPFnYVKDRVDtVDHTtFKYNYSVIaGGPIGDTLEsISNEIvIVATPnGGSILKISNKYHTiGDHEVKAEQVeASKEMGnTLLRAVESYLLAHSDAYNn Bet v 1 (“3005-27”):Y5V, N28T, K320, E45S, N78K, K97S, K103V, K134E, +160N, T10K, K65N,E141N, K1231, D1O9N, E42S, E73T, E87A, K119N, A13OV, V2L, E8S, N47K,D156H, E6S. (SEQ ID NO 15)GIFNvsTsTpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKISFPEGLPFnYVKDRVDtVDHTtFKYNYSVIaGGPIGDTLEsISNEIvIVATPnGGSILKISNKYHTiGDHEVKAEQVeASKEMGnTLLRAVESYLLAHShAYNn Bet v 1 (“3007-6”): Y5V,N28T, K32S, E45S, N78K, K97S K103V, P108G, D125Y, K134E, +160N, E87A,E141N, K55N, N47K, S155T.GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnI (SEQ ID NO 16)SFPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHtDAYNn Bet v 1 (“3007-10”):Y5V, N2BT, K325, E455, N78K, K97S K103V, P108G, D125Y, K134E, +160N,E87A, E141N, K55N, N47K, 5155T, AI3OV, EBS, E73T, V2L.GIFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS (SEQ ID NO 17)FPEGLPFKYVKDRVDtVDHTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHtDAYNn Bet v 1 (“3007-17”):Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, P108G, D125Y, K134E, +160N,K65N, T10P E87A, D156H, EI41N, E42S.GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK (SEQ ID NO 18)ISFPEGLPFnYVkDRVDEVDHIkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHShAYNn Bet v 1 (“3007-22”):Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, P108G, D125Y, K134E, +160N,K65N, T10P E87A, D156H, E141N, E42S, A130V, E8S, N47K, H76T, V2L.GIFNvETsTpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKIS (SEQ ID NO 19)FPEGLPFnYVKDRVDEVDtTkFKYNYSVIaGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHShAYNn Bet v 1 (“3008-8”):Y5V, N28T, K32Q, E455, E73S, E965, P108G, D125Y, N159G, +160N, K134E,N78K, E87A, KI19N, E85, K55N, E141N, N47K.GVFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnI (SEQ ID NO 20)SFPEGLPFKYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDGGSILKISNnYHTKGyHEVKAEQVeASKEMGnTLLRAVESYLLAHSDAYgn Bet v 1 (“3008-13”):Y5V, N28T, K32Q, E455, E735, E965, P108G, D125Y, N159G, +160N, K134E,N78K, E87A, K119N, E85, K55N, E141N, N47K, 5156T, E65, K103V, A130V,V2L. GIFNvsTsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS (SEQ IDNO 21) FPEGLPFKYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGGSILKISNnYHTKGyHEVKvEQVeASKEMGnTLLRAVESYLLAHtDAYgn Bet v 1 (“3008-20”):Y5V, N28T, K32Q, E455, E735, E96S, P108G, D125Y, N159G, +160N, K65N,T10P, E138N, E87A, E425, D156H, N78K.GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK (SEQ ID NO 22)ISFPEGLPFnYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDGGSILKISNKYHTKGyHEVKAEQVKASKnMGETLLRAVESYLLAHShAYgn Bet v 1 “3008-25”):Y5V, N28T, K32Q, E45S, E73S, E96S, P108G, D125Y, N159G, +160N, K65N,T10P, E138N, E87A, E42S, D156H, N78K, KI19N, N47K, T77A, E130V, K115N.GIFNvsTETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKKI (SEQ ID NO 23)SFPEGLPFnYVKDRVDsVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGGSILKISNKYHTKGyHEVKvEQVKASKnMGETLLRAVESYLLAHthAYgn Bet v 1 (“3009-9”):Y5V, N28T, K32Q, E45S, E96S, P108G, +160N, K134E, N78K, E87A, K119N,E8S, K55N, E138N, E141N, S155T, N47K, E6S, K103V, A130V, V2L, R70N,D125Y. GVFNvETsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSyENIsGRGGPGTIKnI (SEQ IDNO 24) SFPEGLPFKYVKDRVDEVDHTkFKYNYSVIaGGPIGDTLsKISNEIKIVATgDGGSILKISNnYHTKGDHEVKAEQVeASKnMGnTLLRAVESYLLAHtDAYNn Bet v 1 (“3009-15”):Y5V, N28T, K320, E45S, E96S, P108G, +160N, K134E, N78K, E87A, K119N,E8S, K55N, E138N, E141N, S155T, N47K, E6S, K103V, A130V, V2L, R70N,D125Y. GIFNvsTsTTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGkGGPGTIKnIS (SEQ IDNO 25) FPEGLPFKYVKDnVDEVDHTkFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGGSILKISNnYHTKGyHEVKvEQVeASKnMGnTLLRAVESYLLAHtDAYNn Bet v 1 (“3009-22”):Y5V, N28T, K32Q, E45S, E96S, P108G, +160N, T77A, K103V, E138N, K65N,T10P, D125Y, E42S.GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK (SEQ ID NO 26)ISFPEGLPFnYVKDRVDEVDHaNFKYNYSVIEGGPIGDTLsKISNEIvIVATgDGGSILKISNKYHTKGyHEVKAEQVKASKnMGETLLRAVESYLLAHSDAYNn Bet v 1 (“3009-28”):Y5V, N28T, K32Q, E45S, E96S, P108G, +160N, T77A, K103V, E138N, K65N,T10P, D125Y, D156H, KI19N E87A, E42S, A130V.GVFNvETETpSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNISGNGGPGTIKK (SEQ ID NO 27)ISFPEGLPFnYVKDRVDEVDHaNFKYNYSVIaGGPIGDTLsKISNEIvIVATgDGGSILKISNnYHTKGyHEVKvEQVKASKnMGETLLRAVESYLLAHShAYNn Bet v 1 clone(“3031”): GVFNVETETASVIPAARLFNAFILDGDTLFPQVAPQAISSVSNISGNGGPGTI (SEQ IDNO 28) KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIEGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN N Bet v 1 clone(“3032”): GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI (SEQ IDNO 29) KKISFPEGLPFNYVKDRVDPVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN N Bet v 1 clone(“3033”): GVFNVETETPSVIPAARLFHAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI (SEQ IDNO 30) KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN N Bet v 1 clone(“3034”): GVFNVETETTSVIPAARLFHAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 31) KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N Bet v 1 clone(“3035”): GVFNVETETPSVIPAARLFMAFILDGDTLFPQVAPPAISSVSNISGNGGPGTI (SEQ IDNO 32) KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAYN N Bet v 1 clone(“3036”): GVFNVETETPSVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 33) KKISFPEGLPFNYVKDRVDTVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N Bet v 1 clone(“3037”): GVFNVETETPSVIPAARLFQAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 34) KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN N Bet v I clone(“3038”): GVFNVETETASVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 35) KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N Bet v 1 clone(“3039”): GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPEAISSVSNISGNGGPGTI (SEQ IDNO 36) KKISFPEGLPFNYVKDRVDGVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAYLLAHSHAY NN Bet v 1 clone(“3040”): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 37) KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAYN N Bet v 1 clone“3041”): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 38) KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N Bet v 1 clone(“3042”): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 39) KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N Bet v 1 clone(“3043”): GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI (SEQ IDNO 40) KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN N Bet v 1 clone(“3044”): GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI (SEQ IDNO 41) KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAY NN Bet v 1 clone(“3045”): GVFNVETETPSVIPAARLFMAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI (SEQ IDNO 42) KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN N Bet v 1 (3010):Y5V, N28T, K32Q, E45S, K97S, P108G, +160N, E605, T10N, K103V, K65N,K129N, D125Y, E42S, S149T.GVFNvETETnSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGNGGPGTIKK (SEQ ID NO 43)ISFPsGLPFnYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVflAEQVKASKEMGETLLRAVEtYLLAHSDAYNn Bet v 1 (3011): Y5V,N28T, K32Q, E455, K97S, P108G, +160N, E6OS, T10N, K103V, K65N, K129N,D125Y, E425, 5149T, K134E, N47K, T77N, V2L.GIFNvETETnSVIPAARLFKAFILDGDtLFPqVAPQAISSVsNIsGkGGPGTIKKI (SEQ ID NO 44)SFPsGLPFnYVKDRVDEVDHnNFKYNYSVIEGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGYHEVnAEQVeASKEMGETLLRAVEtYLLAHSDAYNn Bet v 1 (3012): Y5V,N28T, K320, E45S, K97S, P108G, +160N, E60S, T10N, K103V, K65N, K129N,0125Y, E42S, S149T, K134E, N47K, T77N, V2L, E87A, A16G, Q36N, E73S,D93S. GIFNvETETnSVIPAgRLFKAFILDGDtLFPqVAPnAISSVsNISGkGGPGTIKKIS (SEQ IDNO 45) FPsGLpFnyVKDRVDsVDHnNFKYNYSVIaGGPIGsTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVnAEQVeASKEMGETLLRAVEtYLLAHSDAYNn

[0226] Diagnostic Assay

[0227] Furthermore, the recombinant mutant allergens according to theinvention have diagnostic possibilities and advantages. Prior artallergy vaccines are based on extracts of the naturally occurringallergen source, and thus represent a wide variety of isoforms. Theallergic individual has initially been sensitised and has IgE to one orsome of the isoforms present. Some of the isoforms may be relevant withrespect to the allergic reactions of the allergic individual due tohomology and subsequent cross-reactivity with the isoform to which theindividual is allergic, whereas other isoforms may be irrelevant as theydo not harbour any of the IgE binding epitopes to which the allergicindividual has specific IgE. Due to this heterogeneity of thespecificities of the IgE population, some isoforms may therefore be safeto administer, i.e. they do not result in an allergic response via IgE,whereas other isoforms may be harmful causing undesirable side-effects.

[0228] Thus, the mutants of the invention and the compositions of theinvention intended to be administered therapeutically may also be usedfor an in vivo or in vitro diagnostic assay to monitor the relevance,safety or outcome of a treatment with such mutants or compositions.Diagnostic samples to be applied include body samples, such as blood orsera.

[0229] Thus, the invention also relates to a diagnostic assay forassessing relevance, safety or outcome of therapy of a subject using arecombinant mutant allergen according to the invention or a compositionaccording to the invention, wherein an IgE containing sample of thesubject is mixed with said mutant or said composition and assessed forthe level of reactivity between the IgE in said sample and said mutant.The assessing of the level of reactivity between the IgE in the sampleand the mutant may be carried out using any known immunoassay.

[0230] The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0231] This Example describes characterisation of recombinant mutant Betv 1 mutant allergens with diminished IgE-binding affinity. The specificmutant allergens are also disclosed in PCT/DK 01/00764. The followingrepresents an illustrating example of how to prepare mutants accordingto the present invention.

[0232] Identification of Common Epitopes within Fagales Pollen Allergens

[0233] The major birch pollen allergen Bet v 1 shows about 90% aminoacid sequence identity with major allergens from pollens oftaxonomically related trees, i.e Fagales (for instance hazel andhornbeam) and birch pollen allergic patients often show clinicalsymptoms of allergic cross-reactivity towards these Bet v 1 homologousproteins.

[0234] Bet v 1 also shows about 50-60% sequence identity with allergicproteins present in certain fruits (for instance apple and cherry) andvegetables (for instance celery and carrot) and there are clinicalevidence for allergic cross-reactivity between Bet v 1 and these foodrelated proteins.

[0235] In addition, Bet v 1 shares significant sequence identity (20-40%) with a group of plant proteins called pathogenesis-related proteins(PR-10), however there are no reports of allergic cross-reactivitytowards these PR-10 proteins.

[0236] Molecular modelling suggests that the structures of Fagales andfood allergens and PR-10 proteins are close to being identical with theBet v 1 structure.

[0237] The structural basis for allergic Bet v 1 cross-reactivity wasreported in (Gajhede et al 1996, ref. 17). Thus, any IgE recognisingepitopes on Bet v 1 would be able to cross-react and bind to otherFagales major pollen allergens and give rise to allergic symptoms.

[0238] Selection of Amino Acid Residues for Site-Directed Mutagenesis

[0239] Amino acid residues for site-directed mutagenesis were selectedamong surface exposed residues present in Bet v 1. The relativeorientation and percentage of solvent-exposure of each amino acidresidue was calculated based on their atomic coordinates. Residueshaving a low degree of solvent exposure (<20% ) were not regardedrelevant for mutagenesis due to the possible disruption of the structureor lack of antibody interaction. The remaining residues were rankedaccording to their degree of solvent-exposure.

[0240] Sequence Alignment

[0241] Sequences homologous to the query sequence (Bet v 1 No. 2801, WHOIUIS Nomenclature Subcommittee on Allergens) were derived from GenBankand EMBL sequence databases by a BLAST search (Altschul et al., ref.18). All sequences with BLAST reported probabilities less than 0.1 weretaken into consideration and one list were constructed containing anon-redundant list of homologous sequences. These were aligned byCLUSTAL W (Higgins et al., ref. 19) and the percentage identity werecalculated for each position in the sequence considering the completelist or taxonomically related species only. A total of 122 sequenceswere homologous to Bet v 1 No. 2801 of which 57 sequences originatesfrom taxonomically related species.

[0242] Cloning of the Gene Encoding Bet v 1

[0243] RNA was prepared from Betula verrucosa pollen (Allergon, Sweden)by phenol extraction and LiCl precipitation. Oligo(dT)-celluloseaffinity chromatography was performed batch-wise in Eppendorph tubes,and double-stranded cDNA was synthesised using a commercially availablekit (Amersham). DNA encoding Bet v 1 was amplified by PCR and cloned. Inbrief, PCR was performed using cDNA as template, and primers designed tomatch the sequence of the cDNA in positions corresponding to the aminoterminus of Bet v 1 and the 3′-untranslated region, respectively. Theprimers were extended in the 5′-ends to accommodate restriction sites(Ncol and Hindlll) for directional cloning into pKK233-2.

[0244] Subcloning into pMAL-c

[0245] The gene encoding Bet v 1 was subsequently subcloned into themaltose binding protein fusion vector pMAL-c (New England Biolabs). Thegene was amplified by PCR and subcloned in frame with malE to generatemaltose binding protein (MBP)-Bet v 1 protein fusion operons in whichMBP and Bet v 1 were separated by a factor X_(a) protease clevage sitepositioned to restore the authentic aminoterminal sequence of Bet v 1upon cleavage, as described in ref. 15. In brief, PCR was performedusing pKK233-3 with Bet v 1 inserted as template and primerscorresponding to the amino- and carboxyterminus of the protein,respectively. The promoter proximal primer was extended in the 5′-end toaccommodate 4 codons encoding an in frame factor X_(a) protease cleavagesite. Both primers were furthermore extended in the 5′-ends toaccommodate restriction sites (Kpnl) for cloning. The Bet v 1 encodinggenes were subcloned using 20 cycles of PCR to reduce the frequency ofPCR artefacts.

[0246] In Vitro Mutagenesis

[0247] In vitro mutagenesis was performed by PCR using recombinantpMAL-c with Bet v 1 inserted as template. Each mutant Bet v 1 gene wasgenerated by 3 PCR reactions using 4 primers. The following examples ofmutants are according to prior art PCT/DK 01/00764. Mutants according tothe invention can be prepared and assayed in a similar fashion.

[0248] Two mutation-specific oligonucleotide primers were synthesisedaccommodating each mutation, one for each DNA strand, see FIGS. 3 and 4.Using the mutated nucleotide(s) as starting point both primers wereextended 7 nucleotides in the 5′-end and 15 nucleotides in the 3′-end.The extending nucleotides were identical in sequence to the Bet v 1 genein the actual region.

[0249] Two generally applicable primers (denoted “all-sense” and “allnon-sense” in FIG. 4) were furthermore synthesised and used for allmutants. These primers were 15 nucleotides in length and correspond insequence to regions of the pMAL-c vector approximately 1 kilobaseupstream and downstream from the Bet v 1. The sequence of the upstreamprimer is derived from the sense strand and the sequence of thedownstream primer is derived from the non-sense strand, see FIG. 4.

[0250] Two independent PCR reactions were performed essentiallyaccording to standard procedures (Saiki et al 1988, ref. 20) with theexception that only 20 temperature cycles were performed in order toreduce the frequency of PCR artefacts. Each PCR reaction used pMAL-cwith Bet v 1 inserted as template and one mutation-specific and onegenerally applicable primer in meaningful combinations.

[0251] Introduction of the four amino acid substitutions (Asn28Thr,Lys32Gln, Glu45Ser, Pro108Gly) in the mutant were performed likedescribed above in a step by step process. First the Glu45Ser mutationthen the Pro108Gly mutation and last the Asn28Thr, and Lys32Glnmutations were introduced using pMAL-c with inserted Bet v 1 No. 2801,Bet v 1 (Glu45Ser), Bet v 1 (Glu45Ser, Pro108Gly) as templates,respectively.

[0252] The PCR products were purified by agarose gel electrophoresis andelectro-elution followed by ethanol precipitation. A third PCR reactionwas performed using the combined PCR products from the first two PCRreactions as template and both generally applicable primers. Again, 20cycles of standard PCR were used. The PCR product was purified byagarose gel electrophoresis and electro-elution followed by ethanolprecipitation, cut with restriction enzymes (BslWI/EcoRI), and ligateddirectionally into pMAL-c with Bet v 1 inserted restricted with the sameenzymes.

[0253]FIG. 5 shows an overview of all 9 Bet v 1 mutations, which are asfollows

[0254] Thr10Pro, Asp25Gly, Asn28Thr +Lys32Gln, Glu45Ser, Asn47Ser,Lys55Asn, Glu60Ser, Thr77Ala and Pro108Gly. An additional mutant withfour mutations was also prepared (Asn28Thr, Lys32Gln, Glu45Ser,Pro108Gly). Of these, five mutants were selected for further testing:Asn28Thr+Lys32Gln, Glu45Ser, Glu60Ser, Pro108Gly and the Asn28Thr,Lys32Gln, Glu45Ser, Pro108Gly mutant.

[0255] Nucleotide Sequencing

[0256] Determination of the nucleotide sequence of the Bet v 1 encodinggene was performed before and after subcloning, and following in vitromutagenesis, respectively.

[0257] Plasmid DNA's from 10 ml of bacterial culture grown to saturationovernight in LB medium supplemented with 0.1 g/l ampicillin werepurified on Qiagen-tip 20 columns and sequenced using the Sequenaseversion 2.0 DNA sequencing kit (USB) following the recommendations ofthe suppliers.

[0258] Expression and Purification of Recombinant Bet v 1 and Mutants

[0259] Recombinant Bet v 1 (Bet v 1 No. 2801 and mutants) wereover-expressed in Escherichia coli DH 5a fused to maltose-bindingprotein and purified as described in ref. 15. Briefly, recombinantE.coli cells were grown at 37° C. to an optical density of 1.0 at 436nm, whereupon expression of the Bet v 1 fusion protein was induced byaddition of IPTG. Cells were harvested by centrifugation 3 hourspost-induction, re-suspended in lysis buffer and broken by sonication.After sonication and additional centrifugation, recombinant fusionprotein was isolated by amylose affinity chromatography and subsequentlycleaved by incubation with Factor Xa (ref. 15). After F Xa cleavage,recombinant Bet v 1 was isolated by gelfiltration and if foundnecessary, subjected to another round of amylose affinity chromatographyin order to remove trace amounts of maltose-binding protein.

[0260] Purified recombinant Bet v 1 was concentrated by ultrafiltrationto about 5 mg/ml and stored at 4° C. The final yields of the purifiedrecombinant Bet v 1 preparations were between 2-5 mg per litre E. colicell culture.

[0261] The purified recombinant Bet v 1 preparations appeared as singlebands after silver-stained SDS-polyacrylamide electrophoresis with anapparent molecular weight of 17.5 kDa. N-terminal sequencing showed theexpected sequences as derived from the cDNA nucleotide sequences andquantitative amino acid analysis showed the expected amino acidcompositions.

[0262] We have previously shown (ref. 15) that recombinant Bet v 1 No.2801 is immunochemically indistinguishable from naturally occurring Betv 1.

[0263] Immunoelectrophoresis using Rabbit Polyclonal Antibodies

[0264] The seven mutant Bet v 1 were produced as recombinant Bet v 1proteins and purified as described above and tested for their reactivitytowards polyclonal rabbit antibodies raised against Bet v 1 isolatedfrom birch pollen. When analysed by immunoelectrophoresis (rocket-lineimmunoelectrophoresis) under native conditions, the rabbit antibodieswere able to precipitate all mutants, indicating that the mutants hadconserved α-carbon backbone tertiary structure.

[0265] In order to analyse the effect on human polyclonal IgE-response,the mutants Glu45Ser, Pro108Gly, Asn28Thr+Lys32Gln and Glu60Ser wereselected for further analysis.

[0266] Bet v 1 Glu45Ser Mutant

[0267] Glutamic acid in position 45 show a high degree ofsolvent-exposure (40% ). A serine residue was found to occupy position45 in some of the Bet v 1 homologous PR-10 proteins arguing for thatglutamic acid can be replaced by serine without distortion of theα-carbon backbone tertiary structure. In addition, as none of the knownFagales allergen sequences have serine in position 45, the substitutionof glutamic acid with serine gives rise to a non-naturally occurring Betv 1 molecule.

[0268] T Cell Proliferation Assay using Recombinant Glu45Ser Bet v 1Mutant

[0269] The analysis was carried out as described in Spangfort et al1996a. It was found that recombinant Bet v 1 Glu45Ser mutant was able toinduce proliferation in T cell lines from three different birch pollenallergic patients with stimulation indices similar to recombinant andnaturally occurring.

[0270] Crystallisation and Structural Determination of RecombinantGlu45Ser Bet v 1

[0271] Crystals of recombinant Glu45Ser Bet v 1 were grown by vapourdiffusion at 25° C., essentially as described in (Spangfort et al 1996b,ref. 21). Glu45Ser Bet v 1, at a concentration of 5 mg/ml, was mixedwith an equal volume of 2.0 M ammonium sulphate, 0.1 M sodium citrate,1% (v/v) dioxane, pH 6.0 and equilibrated against 100× volume of 2.0 Mammonium sulfate, 0.1 M sodium citrate, 1% (v/v) dioxane, pH 6.0. After24 hours of equilibration, crystal growth was induced by applying theseeding technique described in ref. 21, using crystals of recombinantwild-type Bet v 1 as a source of seeds.

[0272] After about 2 months, crystals were harvested and analysed usingX-rays generated from a Rigaku rotating anode as described in ref. 21and the structure was solved using molecular replacement.

[0273] Structure of Bet v 1 Glu45Ser Mutant

[0274] The structural effect of the mutation was addressed by growingthree-dimensional Bet v 1 Glu45Ser protein crystals diffracting to 3.0 Åresolution when analysed by X-rays generated from a rotating anode. Thesubstitution of glutamic acid to serine in position 45 was verified bythe Bet v 1 Glu45Ser structure electron density map which also showedthat the overall α-carbon backbone tertiary structure is preserved.

[0275] IgE-Binding Properties of Bet v 1 Glu45Ser Mutant

[0276] The IgE-binding properties of Bet v 1 Glu45Ser mutant wascompared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assayusing a pool of serum IgE derived from birch allergic patients.

[0277] Recombinant Bet v 1 no. 2801 was biotinylated at a molar ratio of1:5 (Bet v 1 no. 2801:biotin). The inhibition assay was performed asfollows: a serum sample (25 μl) was incubated with solid phase anti IgE,washed, re-suspended and further incubated with a mixture ofbiotinylated Bet v 1 no. 2801 (3.4 nM) and a given mutant (0-28.6 nM).The amount of biotinylated Bet v 1 no. 2801 bound to the solid phase wasestimated from the measured RLU after incubation with acridinium esterlabelled streptavidin. The degree of inhibition was calculated as theratio between the RLU's obtained using buffer and mutant as inhibitor.

[0278]FIG. 6 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant.

[0279] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 reaches 50%inhibition at about 6.5 ng whereas the corresponding concentration forBet v 1 Glu45Ser mutant is about 12 ng. This show that the pointmutation introduced in Bet v 1 Glu45Ser mutant lowers the affinity forspecific serum IgE by a factor of about 2.

[0280] The maximum level of inhibition reached by the Bet v 1 Glu45Sermutant is clearly lower compared to recombinant Bet v 1. This mayindicate that after the Glu45Ser substitution, some of the specific IgEpresent in the serum pool are unable to recognise the Bet v 1 Glu45Sermutant.

[0281] Bet v 1 Mutant Asn28Thr+Lys32Gln

[0282] Aspartate and lysine in positions 28 and 32, respectively show ahigh degree of solvent-exposure (35% and 50%, respectively). In thestructure, aspartate 28 and lysine 32 are located close to each other onthe molecular surface and most likely interact via hydrogen bonds. Athreonine and a gluatamate residue were found to occupy positions 28 and32, respectively in some of the Bet v 1 homologous PR-10 proteinsarguing for that aspartate and lysine can be replaced with threonine andglutamate, respectively without distortion of the α-carbon backbonetertiary structure. In addition, as none of the naturally occurringisoallergen sequences have threonine and glutamate in positions 28 and32, respectively, the substitutions gives rise to a non-naturallyoccurring Bet v 1 molecule.

[0283] IgE-Binding Properties of Bet v 1 Mutant Asn28Thr+Lys32Gln

[0284] The IgE-binding properties of mutant Asn28Thr+Lys32Gln wascompared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assayusing the pool of serum IgE derived from birch allergic patientsdescribed above.

[0285]FIG. 7 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 mutant Asn28Thr+Lys32Gln.

[0286] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 reaches 50%inhibition at about 6.5 ng whereas the corresponding concentration forBet v 1 mutant Asn28Thr+Lys32Gln is about 12 ng. This show that thepoint mutations introduced in Bet v 1 mutant Asn28Thr+Lys32Gln lowersthe affinity for specific serum IgE by a factor of about 2.

[0287] The maximum level of inhibition reached by the Bet v 1 mutantAsn28Thr+Lys32Gln mutant is clearly lower compared to recombinant Betv 1. This may indicate that after the Asn28Thr+Lys32Gln substitutions,some of the specific IgE present in the serum pool are unable torecognise the Bet v 1 mutant Asn28Thr+Lys32Gln.

[0288] Bet v 1 Mutant Pro108Gly

[0289] Proline in position 108 shows a high degree of solvent-exposure(60%). A glycine residue was found to occupy position 108 in some of theBet v 1 homologous PR-10 proteins arguing for that proline can bereplaced with glycine without distortion of the α-carbon backbonetertiary structure. In addition, as none of the naturally occurringisoallergen sequences have glycine in position 108, the substitution ofproline with glycine gives rise to a non-naturally occurring Bet v 1molecule.

[0290] IgE-Binding Properties of Bet v 1 Pro108Gly Mutant

[0291] The IgE-binding properties of Bet v 1 Pro108Gly mutant wascompared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assayusing the pool of serum IgE derived from birch allergic patientsdescribed above.

[0292]FIG. 8 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Pro108Gly mutant.

[0293] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 reaches 50%inhibition at about 6.5 ng whereas the corresponding concentration forBet v 1 Pro108Gly is 15 ng. This show that the single point mutationintroduced in Bet v 1 Pro108Gly lowers the affinity for specific serumIgE by a factor of about 2.

[0294] The maximum level of inhibition reached by the Bet v 1 Pro108Glymutant is somewhat lower compared to recombinant Bet v 1. This mayindicate that after the Pro108Gly substitution, some of the specific IgEpresent in the serum pool are unable to recognise the Bet v 1 Pro108Glymutant.

[0295] Bet v 1 Mutant Glu60Ser Mutant

[0296] Glutamic acid in position 60 show a high degree ofsolvent-exposure (60%). A serine residue was found to occupy position 60in some of the Bet v 1 homologous PR-10 proteins arguing for thatglutamic acid can be replaced with serine without distortion of theα-carbon backbone tertiary structure. In addition, as none of thenaturally occurring isoallergen sequences have serine in position 60,the substitution of glutamic acid with serine gives rise to anon-naturally occurring Bet v 1 molecule.

[0297] IgE-Binding Properties of Bet v 1 Glu60Ser Mutant

[0298] The IgE-binding properties of Bet v 1 Glu60Ser mutant wascompared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assayusing the pool of serum IgE derived from birch allergic patientsdescribed above.

[0299]FIG. 9 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu60Ser mutant. In contrast tothe Glu45Ser, Pro108Gly and Asn28Thr+Lys32Gln mutants, the substitutionglutamic acid 60 to serine, does not shown any significant effect on theIgE-binding properties of.

[0300] Structural Analysis of Bet v 1 Glu45Ser, Asn28Thr+Lys32Gln andPro108Gly Mutant

[0301] The structural integrity of the purified recombinant protein wasanalysed by circular dichroism (CD) spectroscopy. FIG. 10 shows the CDspectra of recombinant mutant and recombinant naturally occurringprotein, recorded at close to equal concentrations. The overlap in peakamplitudes and positions in the CD spectra from the two recombinantproteins shows that the two preparations contain equal amounts ofsecondary structures strongly suggesting that the α-carbon backbonetertiary structure is not affected by the introduced amino acidsubstitutions.

[0302] IgE-Binding Properties of Bet v 1 Glu45Ser, Asn28Thr+Lys32Gln andPro108Gly Mutant

[0303] The IgE-binding properties of the mutant was compared withrecombinant Bet v 1 in a fluid-phase IgE-inhibition assay using the poolof serum IgE derived from birch allergic patients described above.

[0304]FIG. 11 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by the Bet v 1 mutant. In contrast to thesingle mutants described above, the inhibition curve of the mutant is nolonger parallel relative to recombinant. This shows that thesubstitutions introduced in the mutant have changed the IgE-bindingproperties and epitope profile compared to recombinant. The lack ofparallellity makes it difficult to quantify the decrease of the mutantsaffinity for specific serum IgE.

[0305] Recombinant Bet v 1 reaches 50% inhibition at about 6 ng whereasthe corresponding concentration for Bet v 1 (Asn28Thr, Lys32Gln,Glu45Ser, Pro108Gly) mutant is 30 ng, i.e a decrease in affinity by afactor 5. However, in order to reach 80% inhibition the correspondingvalues are 20 ng and 400 ng, respectively, i.e a decrease by a factor20.

[0306] T Cell Proliferation Assay Using the Recombinant Bet v 1Glu45Ser, Asn28Thr+Lys32Gln and Pro108Gly Mutant

[0307] The analysis was carried out as described in ref. 15. It wasfound that recombinant Bet v 1 mutant was able to induce proliferationin T cell lines from three different birch pollen allergic patients withstimulation indices similar to recombinant and naturally occurring. Thissuggests that the mutant can initiate the cellular immune responsenecessary for antibody production.

Example 2

[0308] In Vitro Mutagenesis of Mutants According to the PresentInvention

[0309] In vitro mutagenesis was performed by PCR using recombinantpMAL-c with Bet v 1 inserted as template. Preparation of recombinantmutant allergens included two PCR steps; step I and II. First, eachsingle mutation (or several mutations if located closely together in theDNA sequence) was introduced into sequential DNA sequences of Bet v1.2801 derivatives i.e. Bet v 1 (2595) or Bet v 1 (2628) or Bet v 1(2733) using sense and anti-sense mutation-specific oligonucleotideprimers accommodating each mutation(s) along with sense and anti-senseoligonucleotide primers accommodating either upstream or downstreamneighbour mutations or the N-terminus/C-terminus of Bet v 1,respectively as schematically illustrated in FIG. 12 (I). Secondly, PCRproducts from PCR reaction I were purified, mixed and used as templatesfor an additional PCR reaction (II) with oligonucleotide primersaccommodating the N-terminus and C-terminus of Bet v 1 as schematicallyillustrated in FIG. 13 (II). The PCR products were purified by agarosegel electrophoresis and PCR gel purification (Life Techhnologies)followed by ethanol precipitation, cut with restriction enzymes(SacI/EcoRI) or (SacI/XbaI), and ligated directionally into pMAL-crestricted with the same enzymes.

[0310]FIG. 13 shows synthesised oligonucleotide primers andschematically illustrations for the construction of Bet v 1 mutants. Thefollowing Bet v 1 mutants were cloned and sequenced (sequencing ofnucleic acid molecules is described in Example 1): Bet v 1 (3004):GVFNvETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVsNIEGNGGPGTIK (SEQ ID NO 6)KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn Bet v 1 (3005):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIK (SEQ ID NO 7)KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn Bet v 1 (3007):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIK (SEQ ID NO 8)KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATgDGGSILKISNKYHTKGyHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYNn Bet v 1 (3009):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIK (SEQ ID NO 9)KISFPEGfPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYN n Bet v 1 (3006):GVFNvETETTSVIPAARLFKAFILDGDtLFPqVAPQAISSVENIsGNGGPGTIK (SEQ ID NO 10)KISFPEGfPFKYVKDRVDEVDHTkFKYNYSVIEGGPIGDTLEsISNEIvIVATPDGGSILKISNKYHTKGDHEVKAEQVeASKEMGETLLRAVESYLLAHSDAYgn Bet v 1 (3008):GVFNvETETTSVIPAARLFKAFILDGDtLFPkVAPQAISSVENIsGNGGPGTIK (SEQ ID NO 11)KISFPEGfPFKYVKDRVDsVDHTNFKYNYSVIEGGPIGDTLsKISNEIKIVATgDGGSILKISNKYHTKGyHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYgn

[0311] Further Mutants Prepared According to the Present Invention:

[0312] Introduction of multiple point mutations into Bet v 1 maypotentially destabilize the α-carbon backbone folding-pattern of themolecule. Introduction of random amino acid substitutions increases thechances of generating stable mutant Bet v 1 molecules. We thereforegenerated a Bet v 1 mutant library containing Bet v 1 mutants with 17-20point mutations of which amino acid substitutions were randomlysubstituted in 7 positions. The library contained hundreds of differentclones. Fifteen Bet v 1 mutants named Bet v 1 (3031) to (3045) wereobtained from this Bet v 1 mutant library generated using degeneratedoligonucleotide primers. These primers accommodated random substitutionof amino acid residues in the positions T10, K20, Q36, E73, E87, K129and S149 of Bet v 1 (FIGS. 14 and 15). These positions werenon-overlapping with point mutations already introduced into Bet v 1(3002) and Bet v 1 (2595) that were used as DNA templates for the sitedirected mutagenesis PCR reactions illustrated in FIG. 15.

[0313] The cloning procedure was the same as illustrated in FIG. 12except that the primers used in the first PCR round were degenerated incertain positions as indicated in FIG. 15 by letters other than G, C, Tor A. Use of other letters than G, C, T or A indicates that the primerscontain several different nucleotides in these positions. Eight PCRproducts spanning the Bet v 1 gene were produced and purified in thefirst PCR round and then assembled using end-primers (3076s and 3067a)in a second PCR reaction where the eight PCR products from the first PCRround were used as a template.

[0314] The Bet v 1 mutants 3031 to 3045 were DNA sequenced as describedfor the Bet v 1 3004, 3005, 3007 and 3007 mutants in order to verify thenumber and nature of the introduced point mutations: Bet v 1 clone(“3031“): (SEQ ID NO 28)GVFNVETETASVIPAARLFNAFILDGDTLFPQVAPQAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIEGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPY LLAHSHAYNN Bet v 1clone (“3032”): (SEQ ID NO 29)GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDPVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGY LLAHSHAYNN Bet v 1clone (“3033”): (SEQ ID NO 30)GVFNVETETPSVIPAARLFHAFILDGDTLFPQVAPKAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGY LLAHSHAYNN Bet v 1clone (“3034”): (SEQ ID NO 31)GVFNVETETTSVIPAARLFHAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERY LLAHSHAYNN Bet v 1clone (“3035”): (SEQ ID NO 32)GVFNVETETPSVIPAARLFMAFILDGDTLFPQVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAY LLAHSHAYNN Bet v 1clone (“3036”): (SEQ ID NO 33)GVFNVETETPSVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDTVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERY LLAHSHAYNN Bet v Iclone (“3037”): (SEQ ID NO 34)GVFNVETETPSVIPAARLFQAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPY LLAHSHAYNN Bet v 1clone (“3038”): (SEQ ID NO 35)GVFNVETETASVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERY LLAHSHAYNN Bet v 1clone (“3039”): (SEQ ID NO 36)GVFNVETETASVIPAARLFLAFILDGDTLFPQVAPEAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDGVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEAY LLAHSHAYNN Bet v Iclone (“3040”): (SEQ ID NO 37)GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETY LLAHSHAYNN Bet v 1clone “3041”): (SEQ ID NO 38)GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERY LLAHSHAYNN Bet v 1clone (“3042”): (SEQ ID NO 39)GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDILESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERY LLAHSHAYNN Bet v 1clone (“3043”): (SEQ ID NO 40)GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPY LLAHSHAYNN Bet v 1clone (“3044”): (SEQ ID NO 41)GVFNVETETPSVIPAARLFLAFILDGDTLFPQVAPKAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETY LLAHSHAYNN Bet v 1clone (“3045”): (SEQ ID NO 42)GVFNVETETPSVIPAARLFMAFILDGDNLFPKVAPPAISSVSNISGNGGPGTIKKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVATPDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGY LLAHSHAYNN

Example 3

[0315] Identification and Selection of Amino Acids for Substitution

[0316] The parameters of solvent accessibility and conservation degreewere used to identify and select surface-exposed amino acids suitablefor substitution for the allergens Bet v 1, Der p 2 and Ves v 5.

[0317] Solvent Accessibility

[0318] Solvent accessibility was calculated using the softwareInsightII, version 97.0 (MSI) and a probe radius of 1.4 Å (Connollysurface).

[0319] Internal cavities were excluded from the analyses by filling withprobes using the software PASS (Putative Active Sites with Spheres).Probes on the surface were subsequently removed manually.

[0320] Conservation

[0321] Bet v 1:

[0322] 3-D structure is based on accession number Z80104 (1bv1.pdb).

[0323] 38 other Bet v 1 sequences included in the analysis of conservedresidues comprise accession numbers:

[0324] P15494=X15877=Z80106, Z80101, AJ002107, Z72429, AJ002108, Z80105,Z80100, Z80103, AJ001555, Z80102, AJ002110, Z72436, P43183=X77271,Z72430, AJ002106, P43178=X77267, P43179=X77268, P43177=X77266, Z72438,P43180=X77269, AJ001551, P43185=X77273, AJ001557, Z72434, AJ001556,Z72433=P43186, AJ001554, X81972, Z72431, P45431=X77200, P43184=X77272,P43176=X77265, S47250, S47251, Z72435, Z72439, Z72437,S47249.

[0325] Bet v 1

[0326] 59 amino acids highly solvent exposed:

[0327] K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D-125,R-145, D-109, T-77, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63,H-76, E-8, K-134, E-45, T-10, V-12, K-20, L-62, S-155, H-126, P-50,N-78, K-119, V-2, L-24, E-42, N-4, A-153, I-44, E-138, G-61, A-130,R-70, N-28, P-35, S-149, K-103, Y-150, H-154, N-43, A-106, K-115, P-14,Y-5, K-137, E-141, E-87, E-73.

[0328] 57 amino acids highly solvent exposed and conserved (>70%):

[0329] K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D-125,R-145, D-109, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63, H-76,E-8, K-134, E-45, T-10, V-12, K-20, S-155, H-126, P-50, N-78, K-119,V-2, L-24, E-42, N-4, A-153, I-44, E-138, G-61, A-130, R-70, N-28, P-35,S-149, K-103, Y-150, H-154, N-43, A-106, K-115, P-14, Y-5, K-137, E-141,E-87, E-73.

[0330] Table 1 shows a listing in descending order of solvent exposureof Bet v 1 amino acids. Column 1 lists the amino acid number startingfrom the amino-terminal, column 2 lists the amino acid in one letterabbreviation, column 3 lists the normalised solvent exposure index,column 4 lists the percent of known sequences having the concerned aminoacid in this position. TABLE 1 Bet v 1 NO AA Solv_exp Cons % 129 K 1,00090 60 E 0,986 97 47 N 0,979 100 65 K 0,978 100 108 P 0,929 100 159 N0,869 100 93 D 0,866 100 123 K 0,855 100 32 K 0,855 100 125 D 0,821 74145 R 0,801 90 109 D 0,778 82 77 T 0,775 56 127 E 0,760 100 36 Q 0,74995 131 E 0,725 100 152 L 0,718 97 6 E 0,712 100 96 E 0,696 100 156 D0,693 97 63 P 0,692 97 76 H 0,683 90 8 E 0,638 97 134 K 0,630 100 45 E0,623 100 10 T 0,613 97 12 V 0,592 100 20 K 0,584 100 62 L 0,575 5 155 S0,568 97 126 H 0,551 95 50 P 0,541 100 78 N 0,538 100 119 K 0,529 100 2V 0,528 100 24 L 0,528 100 42 E 0,519 100 4 N 0,517 95 153 A 0,513 10044 I 0,508 97 138 E 0,496 100 61 G 0,488 100 130 A 0,479 97 70 R 0,474100 28 N 0,469 90 35 P 0,467 100 149 S 0,455 92 103 K 0,447 100 150 Y0,438 100 154 H 0,436 100 43 N 0,412 100 106 A 0,411 95 115 K 0,411 10014 P 0,410 97 5 Y 0,410 100 137 K 0,396 100 141 E 0,387 95 87 E 0,385100 73 E 0,384 100 16 A 0,367 100 79 F 0,362 100 3 F 0,355 100 158 Y0,346 100 105 V 0,336 100 101 E 0,326 100 64 F 0,325 100 86 I 0,322 10039 S 0,314 100 124 G 0,310 100 72 D 0,308 97 142 T 0,293 67 66 Y 0,289100 55 K 0,288 100 7 T 0,279 67 40 S 0,274 95 25 D 0,271 87 135 A 0,26792 68 K 0,262 100 97 K 0,247 100 46 G 0,235 100 27 D 0,232 97 1 G 0,227100 113 I 0,225 77 51 G 0,220 100 92 G 0,218 100 80 K 0,212 100 110 G0,211 100 107 T 0,203 85 94 T 0,202 92 41 V 0,201 97 48 G 0,198 100 91 I0,192 18 31 P 0,188 100 75 D 0,188 97 33 V 0,183 100 49 G 0,176 100 17 R0,172 100 99 S 0,158 64 89 G 0,154 100 53 I 0,154 100 121 H 0,153 100 9T 0,150 72 74 V 0,148 97 132 Q 0,146 72 57 S 0,137 49 148 E 0,135 100 82N 0,133 41 128 V 0,125 64 117 S 0,124 87 90 P 0,117 67 116 I 0,112 100122 T 0,107 100 139 M 0,104 62 95 L 0,104 97 54 K 0,096 100 146 A 0,095100 59 P 0,088 97 157 A 0,088 100 133 V 0,077 44 88 G 0,068 100 140 G0,053 85 37 A 0,042 95 81 Y 0,041 100 23 I 0,036 95 104 I 0,036 92 15 A0,036 97 58 F 0,029 100 29 L 0,028 100 19 F 0,027 100 100 N 0,022 97 22F 0,021 97 71 V 0,014 100 111 G 0,014 100 13 I 0,014 100 18 L 0,014 97114 L 0,014 100 11 S 0,007 100 151 L 0,007 97 144 L 0,007 90 52 T 0,007100 84 S 0,007 97 118 N 0,007 97 102 I 0,007 100 21 A 0,000 97 26 G0,000 97 30 F 0,000 44 34 A 0,000 100 38 I 0,000 87 56 I 0,000 100 67 V0,000 97 69 D 0,000 62 83 Y 0,000 95 85 V 0,000 72 98 I 0,000 95 112 S0,000 77 120 Y 0,000 95 136 S 0,000 67 143 L 0,000 100 147 V 0,000 100

Example 4

[0331] This Example describes preparation and characterisation ofrecombinant mutant Bet v 1 allergens with more than four mutations anddiminished IgE-binding affinity according to prior art PCT/DK 01/00764.Mutants according to the present invention are prepared and assayedaccordingly.

[0332] Selection of Amino Acid Residues for Site-Directed Mutagenesis ofBet v 1

[0333] Amino acid residues were selected as described in Example 1.

[0334] In Vitro Mutagenesis

[0335] In vitro mutagenesis was performed by PCR using recombinantpMAL-c with Bet v 1 inserted as template. Preparation of recombinantmutant allergens comprising five to nine primary mutations included twoPCR steps; step I and II. First, each single mutation (or severalmutations if located closely together in the DNA sequence) wasintroduced into sequential DNA sequences of Bet v 1.2801 or Bet v 1.2801derivatives using sense and anti-sense mutation-specific oligonucleotideprimers accommodating each mutation(s) along with sense and anti-senseoligonucleotide primers accommodating either upstream or downstreamneighbour mutations or the N-terminus/C-terminus of Bet v 1,respectively as schematically illustrated in FIG. 15 (I). Secondly, PCRproducts from PCR reaction I were purified, mixed and used as templatesfor an additional PCR reaction (II) with oligonucleotide primersaccommodating the N-terminus and C-terminus of Bet v 1 as schematicallyillustrated in FIG. 15 (II). The PCR products were purified by agarosegel electrophoresis and PCR gel purification (Life Techhnologies)followed by ethanol precipitation, cut with restriction enzymes(SacI/EcoRI) or (SacI/XbaI), and ligated directionally into pMAL-crestricted with the same enzymes.

[0336]FIG. 16 shows synthesised oligonucleotide primers andschematically illustrations for the construction of Bet v 1 mutants withmore than four primary mutations. The mutated amino acids werepreferably selected from the group consisting of amino acids that arecharacterised by being highly solvent exposed and conserved as describedin Example 3. The Bet v 1 mutants are as follows:

[0337] Mutant Bet v 1 (2628): Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser,Lys134Glu.

[0338] Mutant Bet v 1 (2637): Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr,Pro108Gly, Leu152Lys, Ala153Gly, Ser55Pro.

[0339] Mutant Bet v 1 (2733): Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln,Glu45Ser, Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu,Asp156His, +160Asn.

[0340] Mutant Bet v 1 (2744): Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser,Asn78Lys, Lys103Val, Lys123Ile, Asp156His, +160Asn.

[0341] Mutant Bet v 1 (2753): Asn28Thr, Lys32Gln, Lys65Asn, Glu96Leu,Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr, Glu127Ser, Arg145Glu.

[0342] Nucleotide Sequencing and Expression and Purification ofRecombinant Bet v 1 and Mutants

[0343] Sequencing and expression of recombinant protein was performed asdescribed in Example 1.

[0344] Bet v 1 (2628) and Bet v 1 (2637) Mutants

[0345]FIG. 17 shows introduced point mutations at the molecular surfaceof Bet v 1 (2628) and Bet v 1 (2637).

[0346] Crystallisation and Structural Determination of Recombinant Bet v1(2628) Mutant Protein.

[0347] Structural determination was performed as described in Example 1.

[0348] Structure of Bet v 1 (2628) Mutant

[0349] The structural effect of the mutations was addressed by growingthree-dimensional Bet v 1 (2628) protein crystals diffracting to 2.0 Åresolution when analysed by X-rays generated from a rotating anode. Thesubstitutions Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu wereverified by the Bet v 1 (2628) structure electron density map which alsoshowed that the overall α-carbon backbone tertiary structure ispreserved.

[0350] Structural Analysis of Bet v 1 (2637) Mutant

[0351] The structural integrity of the purified Bet v 1 (2637) mutantwas analysed by circular dichroism (CD) spectroscopy. FIG. 18 shows theCD spectra of recombinant Bet v 1.2801 (wildtype) and Bet v 1 (2637)mutant, recorded at close to equal concentrations. The overlap in peakamplitudes and positions in the CD spectra from the two recombinantproteins shows that the two preparations contain equal amounts ofsecondary structures strongly suggesting that the α-carbon backbonetertiary structure is not affected by the introduced amino acidsubstitutions.

[0352] IgE-Binding Properties of Bet v 1 (2628) and Bet v 1 (2637)Mutants.

[0353] The IgE-binding properties of Bet v 1 (2628) and Bet v 1 (2637)as well as a 1:1 mix of Bet v 1 (2628) and Bet v 1 (2637) was comparedwith recombinant wild type Bet v 1.2801 in a fluid-phase IgE-inhibitionassay using a pool of serum IgE derived from birch allergic patients.

[0354] As described in Example 1, recombinant Bet v 1.2801 wasbiotinylated at a molar ratio of 1:5 (Bet v 1 no. 2801:biotin). Theinhibition assay was performed as follows: a serum sample (25 μl) wasincubated with solid phase anti IgE, washed, re-suspended and furtherincubated with a mixture of biotinylated Bet v 1.2801 and a given mutantor 1:1 mix of the two mutants. The amount of biotinylated Bet v 1.2801bound to the solid phase was estimated from the measured RLU afterincubation with acridinium ester labelled streptavidin. The degree ofinhibition was calculated as the ratio between the RLU's obtained usingbuffer and mutant as inhibitor.

[0355]FIG. 19 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1.2801 to serum IgE from a pool of allergic patientsby non-biotinylated Bet v 1.2801 and by Bet v 1 (2628), Bet v 1 (2637)and a 1:1 mix of Bet v 1 (2628) and Bet v 1 (2637).

[0356] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1.2801 reaches50% inhibition at about 5 ng whereas the corresponding concentration forBet v 1 (2628) mutant is about 15-20 ng. This show that the pointmutation introduced in the Bet v 1 (2628) mutant lowers the affinity forspecific serum IgE by a factor of about 3-4.

[0357] The maximum level of inhibition reached by the Bet v 1 (2628)mutant protein is clearly lower compared to recombinant Bet v 1.2801.This may indicate that some of the specific IgE present in the serumpool are unable to recognise the Bet v 1 (2628) mutant protein due tothe introduced point mutations.

[0358] Bet v 1 (2637) reaches 50% inhibition at about 400-500 ng showingthat the point mutation introduced in the Bet v 1 (2637) mutant lowersthe affinity for specific serum IgE by 80 to 100-fold compared to Bet v1.2801. The large difference in IgE-binding is further supported by aclear difference in inclination of the inhibition curve obtained withBet v 1(2637) mutant protein compared to the inhibition curve for Bet v1.2801. The different inclination provide evidence that the reduction inIgE-binding is due to a distinctly different epitope pattern of themutant compared to Bet v 1.2801.

[0359] In addition to the inhibition assays with single modifiedallergens a 1:1 mix of Bet 1 (2628) and Bet v 1 (2637) having same molarconcentration of Bet v 1 as each of the samples with Bet 1 (2628) or Betv 1 (2637), respectively was tested and showed full (100%) capacity toinhibit IgE-binding to rBet v 1.2801. The capacity to fully inhibitIgE-binding is a clear indication that all reactive epitopes present onBet v 1.2801 were present in the 1:1 allergen mix. Further support comesfrom the comparable inclination of the two inhibition curves for Bet v1.2801 and the allergen mix. Reduced IgE-reactivity of the mixedallergen sample is demonstrated by the need of a four-fold higherconcentration of the allergen mix, when compared to Bet v 1.2801, forobtaining 50% inhibition of IgE-binding.

[0360] T Cell Proliferation Assay using Mutated Recombinant Bet v 1Allergens.

[0361] The analysis was carried out as described in ref. 15. Bet v 1(2628) and Bet v 1(2637) mutant protein were both able to induceproliferation in T cell lines from birch pollen allergic patients withstimulation indices similar to recombinant and naturally occurring. Thissuggests that both of Bet v 1 (2628) and Bet v 1 (2637) mutant proteincan each initiate the cellular immune response necessary for antibodyproduction.

[0362] Histamine Release Assays with Human Basophil.

[0363] Histamine release from basophil leucocytes was performed asfollows. Heparinized blood (20 ml) was drawn from each birch pollenpatient, stored at room temperature, and used within 24 hours.Twenty-five microlitres of heparinized whole blood was applied to glassfibre coated microtitre wells (Reference Laboratory, Copenhagen,Denmark) and incubated with 25 microlitres of allergen or anti-IgE for 1hour at 37° C. Thereafter the plates were rinsed and interferingsubstances were removed. Finally, histamine bound to the microfibres wasmeasured spectrophotofluometrically.

[0364] Histamine Release Properties of Bet v 1 (2628) and Bet v 1 (2637)Mutant Protein.

[0365] Histamine release data is shown in FIG. 20 and FIG. 21. Thepotency of Bet v 1 (2628) and Bet v 1 (2637) mutant protein to inducehistamine release in human basophil from two birch pollen allergicpatients has been tested. In both cases the release curve of the mutatedallergens to induce histamine release is clearly shifted to the rightcompared to the release curve of Bet v 1.2801. The shift indicate thatthe potency of Bet v 1 (2628) and Bet v 1 (2637) is reduced 3 to10-fold.

[0366] Mutant Bet v 1 (2744) and Mutant Bet v 1 (2753)

[0367] Bet v 1 (2744) and Bet v 1 (2753) was likewise constructed foruse as a mixed allergen vaccine. In these mutated allergens pointmutations were distributed in an all surface arranged fashion as shownin FIG. 22 and FIG. 23 and was again designed to affect differentsurface areas in the two molecules, respectively, as shown in FIG. 24.However these modified allergens might individually be used as singleallergen vaccines as well.

[0368] Structural Analysis of Bet v 1 (2744) Mutant Protein

[0369] The structural integrity of the purified Bet v 1 (2744) mutantwas analysed by circular dichroism (CD) spectroscopy. FIG. 25 shows theCD spectra of recombinant Bet v 1.2801 (wildtype) and Bet v 1 (2744)mutant, recorded at close to equal concentrations. The overlap in peakamplitudes and positions in the CD spectra from the two recombinantproteins shows that the two preparations contain equal amounts ofsecondary structures strongly suggesting that the α-carbon backbonetertiary structure is not affected by the introduced amino acidsubstitutions.

[0370] Histamine Release Properties of Bet v 1 (2744)

[0371] Histamine release data from five experiments with basophilleucocytes from five different birch pollen allergic patients is shownin FIG. 26 and FIGS. 27A-D. The potency of Bet v 1 (2744) mutant proteinto induce histamine release in human basophil has been tested. Therelease curves of the mutated allergens are clearly shifted to the rightcompared to the release curve of Bet v 1.2801 indicating that thepotency of Bet v 1 (2744) to release histamine is reduced 3 to 5-fold.

[0372] Mutant Bet v 1 (2733)

[0373] A Mutant Bet v 1 (2733) has been constructed and recombinantlyexpressed. The distribution of point mutations in Bet v 1 (2733) leaveseveral surface areas constituting >400Å² unaltered. FIG. 28 showintroduced point mutations at the molecular surface of Bet v 1 (2733).

Example 5

[0374] This Example describes characterisation of recombinant mutant Betv 1 allergens with more than four mutations and diminished IgE-bindingaffinity according to prior art PCT/DK 01/00764. Mutants according tothe present invention are prepared and assayed accordingly.

[0375] T-Cell Reactivity of Recombinant and Mutant Bet v 1:

[0376] Purpose:

[0377] To investigate an in vitro T-cell response to the mutatedallergens in terms of proliferation and cytokine production.

[0378] Methods:

[0379] PBL (Peripheral blood lymphocytes) from allergic patients wereused in the following investigation.

[0380] Eight bet v 1 specific T-cell lines were established from the PBLwith naturally purified bet v 1 in order to sustain the variety of bet v1 isoforms the T-cells are presented to, as described in a previouslypublished protocol (26).

[0381] Ten PBL and eight T-cell lines were stimulated with birch extract(Bet v), naturally purified bet v 1 (nBet v 1), recombinant Bet v 1(rBet v 1 or wt; 27) and four different mutated forms of rBet v 1(described elsewhere): 2595, 2628, 2637, 2744, 2773. The 2637 mutant waslater found to be partly unfolded and will not be discussed.

[0382] In brief: In a round-bottomed 96 well plate PBL were added in2×10⁵ per well. The different birch samples were added in threedifferent concentrations in quadroplicates and allowed to grow for 6days. At day 6 cell half of volume (100 μl) from each well with thehighest concentration of birch were harvested for cytokine production.Radioactive labelled thymidine was added to the wells. Next day (day 7)the cells were harvested on a filter. Scintilation fluid was added tothe filter and the radioactivity was measured in a scintillationcounter.

[0383] Likewise in a 96 well round-bottomed 96 well plate T-cells wereadded in 3×10⁴ T-cells per well and stimulated with irradiatedautologous PBL (1×10⁵ cells/well) and 3 different concentrations of thedifferent birch samples. After 1 day cells from each well with thehighest concentration birch were harvested for cytokine production.Radioactive labelled thymidine were added to the wells. At day 2 thecells were harvested onto a filter and counted as described for PBL.

[0384] Supernatant from the quadroplicates were pooled and cytokineswere measured using a CBA (cytokine bead array) kit from BectonDickinson.

[0385] Results:

[0386] Ten PBL cultures showed specific stimulation to birch. In generalproliferation of the PBL to the different birch samples were similar,although variations could be seen. In 3 PBL, nBet v 1 stimulatedproliferation better than rBet v 1 and the mutants. The mutant birchsamples stimulated PBL almost identical to rBet v 1 (FIG. 29). FIG. 29shows the Stimulation Index for the above-mentioned Bet v 1preparations. The Stimulation Index (SI) is calculated as proliferation(cpm: count per minute) of the stimulated sample (highest concentration)divided with the proliferation (cpm) of the medium control. PPDdesignates purified protein derivative from mucobacterium tuberculosis,which serves as a positive control.

[0387] Cytokine production was dominated by IFN-gamma and increasedproportionally with PBL proliferation. No signs of a Th1/Th2 shift wereapparent (FIGS. 30-32). FIG. 30 shows a patient with a Th0 profile, FIG.31 a Th1 profile and FIG. 32 a Th2 profile. Cytokine production ismeasured in pg/ml indicated as the bars and the ratio betweenIL-5/IFN-gamma is the lower dashed line (Y-axis to the right).Proliferation is measured in cpm seen on the Y-axis to the right as asolid line measured in cpm. Medium and MBP (maltose bindig protein) areincluded as background controls.

[0388] Eight T-cell lines established on nBet v 1 and all, except one,proliferated equally well to all birch samples. Four T-cell lines weresecreting Th0 like cytokines based on the IL-5 and IFN-gamma ratio(Th2>5, 5>Th0>0.2, 0.2>Th1). Three T-cell lines were secreting Th1cytokines and one T-cell line was secreting Th2 cytokines. TheIL-5/IFN-gamma ratio was not affected by the different birch samples.

[0389] Conclusion:

[0390] All PBL cultures and ⅞ T-cell lines that showed specificstimulation to nBet v 1 did also respond to rBet v 1 and the mutants.These data suggests that for T-cell stimulation a single isoform of Betv 1 or these 4 mutants can substitute for the mixture of individualisoforms found in the natural allergen preparations. Thus, vaccinesbased on recombinant allergens or these 4 mutants will address theexisting Bet v 1 specific T-cell population.

Example 6

[0391] This Example describes characterisation of recombinant mutant Betv 1 allergens with more than four mutations and diminished IgE-bindingaffinity according to prior art PCT/DK 01/00764. Mutants according tothe present invention are be prepared and assayed accordingly.

[0392] Induction of Bet v 1 Specific IgG Antibodies and BlockingAntibodies Following Immunization with Recombinant and Mutant Bet v 1Proteins:

[0393] In this section the term “blocking antibodies” is defined asantibodies, different from human IgE antibodies, that are able to bindto an antigen and prevent the binding of human IgE antibodies to thatantigen.

[0394] The ability of recombinant Bet v1 2227 wild type protein (rBetv 1) and Bet v 1 2595, 2628, 2744 and 2773 mutant proteins to induce Betv 1 specific IgG antibodies and blocking antibodies was tested inimmunization experiments in mice.

[0395] BALB/cA mice (8 in each group) were immunized by intraperitonealinjections with recombinant Bet v1 2227 wild type protein or the fourmutant proteins. The mice were immunized four times with a dose intervalof 14 days. The different proteins were conjugated to 1,25 mg/mlAlhydrogel, (Aluminium Hydroxide gel, 1,3% pH 8.0-8.4, SuperfosBiosector). The mice were immunized with either 1 ug protein/dose or 10ug protein/dose. Blood samples were drawn by orbital bleed at day0,14,35, 21, 49 and 63.

[0396] Specific IgG antibody levels was analyzed by direct ELISA usingrBet v 1 coated microtiterplates and biotinylated rabbit anti mouse IgGantibodies (Jackson) as detection antibody. Immunization withrecombinant Bet v1 2227 wild type protein or the four mutant proteinsinduced a strong r Bet v 1 specific IgG response. This findingdemonstrates that the four mutated proteins are able to induceantibodies that are highly cross reactive to the Bet v 1 2227 wild typeprotein.

[0397] To assess the induction of blocking antibodies, serum samplesfrom birch pollen allergic patients were incubated with paramagneticbeads coated with a monoclonal mouse anti-human IgE antibody. Afterincubation, the beads were washed and resuspended in buffer or dilutedsamples (1:100) of mouse serum from un-immunized mice (control) or miceimmunized as described above. Biotinylated r Bet v 1 was then added tothis mixture of beads and mouse serum antibodies. After incubation, thebeads were washed and bound biotinylated rBet v 1 was detected usingacridinium labeled streptavidine. Incubation of beads with serum fromun-immunized mice did not change the binding of r Bet v 1 to the beads.In contrast, incubation of the beads with serum from mice immunized withthe recombinant Bet v1 2227 wild type protein or the four mutantproteins significantly reduced binding of r Bet v 1 to the beadsdemonstrating the presence of Bet v 1 specific blocking antibodies inthe serum samples. Thus, at day 63 one or more serum samples from allhigh dose (10 ug/dose) immunization groups were able to reduce bindingof r Bet v1 to the beads with more than 80%. These findings demonstratethat the four mutated proteins are able to induce antibodies that canact as Bet v 1 specific blocking antibodies.

Example 7

[0398] This example describes the structural characterization andIgE-binding properties of a mutant according to the invention having 12point mutation. The mutations introduced in mutant 3007 are described inexample 2.

[0399] Structural Analysis of Bet v 1 (3007) Mutant Protein

[0400] The structural integrity of the purified Bet v 1 (3007) mutantwas analysed by circular dichroism spectroscopy as described inexample 1. FIG. 33 shows the CD spectra of recombinant Bet v 1.2801(wildtype) and Bet v 1 (3007) mutant, recorded at equal concentrationsas previously described in example 1. The overlap in amplitude-positionsin the CD spectra from the two recombinant proteins indicates that thetwo preparations contain roughly equal amounts of secondary structures,strongly suggesting that the α-carbon backbone tertiary structure is not or affected by the introduced amino acid substitutions.

[0401] IgE-Binding Analysis of Bet v 1 (3007) Mutant Protein

[0402]FIG. 34 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1.2801 to serum IgE from a pool of allergic patientsby non-biotinylated Bet v 1.2801 (wildtype) and the Bet v 1 (3007)mutant according to methods described in example 4. There is a cleardifference in the amount of the respective recombinant proteinsnecessary to reach 50% inhibition of the binding to serum IgE present inthe serum pool. Recombinant Bet v 1.2801 reaches 50% inhibition at about5 ng whereas the corresponding concentration for Bet v 1 (3007) mutantis about 200 ng. The level of inhibition reached by the Bet v 1 (3007)mutant protein is clearly lower compared to recombinant Bet v 1.2801.This show that the 12 point mutations introduced in the Bet v 1 (3007)mutant lowers the affinity for specific serum IgE.

[0403] References

[0404] 1. WO 97/30150 (Pangenetics B. V., Molecules for the induction ofimmunological tolerance)

[0405] 2. WO 92/02621 (Biomay Biotechnik Produktions—undHandelsgesellschaft mbH, Allergens of Alder pollen and applicationsthereof)

[0406] 3. WO 90/11293 (Immunologic Pharmaceutical Corporation, TheUniversity of North Carolina at Chapel Hill, Allergenic proteins fromragweed and uses thereof)

[0407] 4. Takai T, Yokota T, Yasue M, Nishiyama C, Yuuki T, Mori A,Okudaira H, Okumura Y: “Engineering of the major house dust miteallergen Der f 2 for allergen-specific immunotherapy”. Nat Biotechnol15, 754-758 (1997).

[0408] 5. Smith A M, Chapman M D: “Localization of antigenic sites onDer p 2 using oligonucleotide-directed mutagenesis targeted to predictedsurface residues”. Clin Exp Allergy 27, 593-599 (1997).

[0409] 6. Aki T, Ono K, Hidaka Y, Shimonishi Y, Jyo T, Wada T, YamashitaM, Shigeta S, Murooka Y, Oka S: “Structure of IgE epitopes on a new39-kD allergen molecule from the house dust mite, Dermatophagoidesfarinae”. Int Arch Allergy Immunol 103, 357-364 (1994).

[0410] 7. Forster E, Dudler T, Gmachl M, Aberer W, Urbanek R, Suter M:“Natural and recombinant enzymatically active or inactive bee venomphospholipase A2 has the same potency to release histamine frombasophils in patients with Hymenoptera allergy”. J Allergy Clin Immunol95,1229-1235 (1995).

[0411] 8. Burks A W, Shin D, Cockrell G, Stanley J S, Helm R M, Bannon GA: “Mapping and mutational analysis of the IgE-binding epitopes on Ara h1, a legume vicilin protein and a major allergen in peanuthypersensitivity”. Eur J Biochem 245, 334-339 (1997).

[0412] 9. Stanley J S, King N, Burks A W, Huang S K, Sampson H, CockrellG, Helm R M, West C M, Bannon G A: “Identification and mutationalanalysis of the immunodominant IgE binding epitopes of the major peanutallergen Ara h 2”. Arch Biochem Biophys 342, 244-253 (1997).

[0413] 10. Ferreira F, Rohlfs A, Hoffmann-Sommergruber K, Schenk S,Ebner C, Briza P, Jilek A, Kraft D, Breitenbach M, Scheiner O:“Modulation of IgE-binding properties of tree pollen allergens bysite-directed mutagenesis”. Adv Exp Med Biol 409, 127-135 (1996).

[0414] 11. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl A J,Grimm R, Jah-Schmid B, Breiteneder H, Kraft D, Breitenbach M,Rheinberger H -J, Scheiner O, “Modulation of IgE reactivity of allergensby site-directed mutagenesis: Potential use of hypeallergenic variantsfor immunotherapy”, FASEB Journal for Experimental Biology Vol. 12, No.2, February 1998, 231-242 (1998).

[0415] 12. Wiedemann P, Giehl K, Almo S C, Fedorov A A, Girvin M,Steinberger P, Rudiger M, Ortner M, Sippl M, Dolecek C, Kraft D,Jockusch B, Valenta R: “Molecular and structural analysis of acontinuous birch profilin epitope defined by a monoclonal antibody”. JBiol Chem 271, 29915-29921 (1996).

[0416] 13. Alvarez A M, Fukuhara E, Nakase M, Adachi T, Aoki N, NakamuraR, Matsuda T: “Four rice seed cDNA clones belonging to thealpha-amylase/trypsin inhibitor gene family encode potential riceallergens”. Biosci Biotechnol Biochem 59,1304-1308 (1995).

[0417] 14. Colombo P, Kennedy D, Ramsdale T, Costa M A, Djro G, Izzo V,Salvadori S, Guerrini R, Cocchiara R, Mirisola M G, Wood S, Geraci D,Journal of Immunology Vol.160, No. 6, Mar. 15, 1998, 2780-2875.

[0418] 15. Spangfort M D, Ipsen H, Sparholt S H, Aasmul-Olsen S, LarsenM R, Mortz E, Roepstorff P, Larsen J N: “Characterization of PurifiedRecombinant Bet v 1 with Authentic N-terminus, Cloned in Fusion withMaltose-Binding Protein”. Prot Exp Purification 8, 365-373 (1996a).

[0419] 16. Ipsen H, Wihl J -Å, Petersen B N, Lwenstein H: “Specificitymapping of patients IgE response towards the tree pollen major allergensAln g I, Bet v I and Cor a I.” Clin. Exp. Allergy 22, 391-9, (1992)

[0420] 17. Gajhede M, Osmark P, Poulsen F M, Ipsen H, Larsen J N, Joostvan Neerven R J, Schou C, Lwnstein H, and Spangfort M D: “X-ray and NMRstructure of Bet v 1, the origin of birch pollen allergy”. Naturestructural biology 3, 1040-1045 (1996).

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[0423] 20. Saiki R K, Gelfand D H, Stoffel S, Scharf S J, Higuchi R,Horn G T, Mullis K B, Erlich H A: “Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase”. Science 239,487-491 (1988).

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[0431]

1 89 1 160 PRT Betula verrucosa 1 Met Gly Val Phe Asn Tyr Glu Thr GluThr Thr Ser Val Ile Pro Ala 1 5 10 15 Ala Arg Leu Phe Lys Ala Phe IleLeu Asp Gly Asp Asn Leu Phe Pro 20 25 30 Lys Val Ala Pro Gln Ala Ile SerSer Val Glu Asn Ile Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys LysIle Ser Phe Pro Glu Gly Leu Pro 50 55 60 Phe Lys Tyr Val Lys Asp Arg ValAsp Glu Val Asp His Thr Asn Phe 65 70 75 80 Lys Tyr Asn Tyr Ser Val IleGlu Gly Gly Pro Ile Gly Asp Thr Leu 85 90 95 Glu Lys Ile Ser Asn Glu IleLys Ile Val Ala Thr Pro Asp Gly Gly 100 105 110 Ser Ile Leu Lys Ile SerAsn Lys Tyr His Thr Lys Gly Asp His Glu 115 120 125 Val Lys Ala Glu GlnVal Lys Ala Ser Lys Glu Met Gly Glu Thr Leu 130 135 140 Leu Arg Ala ValGlu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn 145 150 155 160 2 160PRT Betula verrucosa 2 Met Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr SerVal Ile Pro Ala 1 5 10 15 Ala Arg Leu Phe Lys Ala Phe Ile Leu Asp GlyAsp Asn Leu Phe Pro 20 25 30 Lys Val Ala Pro Gln Ala Ile Ser Ser Val GluAsn Ile Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys Lys Ile Ser PhePro Glu Gly Phe Pro 50 55 60 Phe Lys Tyr Val Lys Asp Arg Val Asp Glu ValAsp His Thr Asn Phe 65 70 75 80 Lys Tyr Asn Tyr Ser Val Ile Glu Gly GlyPro Ile Gly Asp Thr Leu 85 90 95 Glu Lys Ile Ser Asn Glu Ile Lys Ile ValAla Thr Pro Asp Gly Gly 100 105 110 Ser Ile Leu Lys Ile Ser Asn Lys TyrHis Thr Lys Gly Asp His Glu 115 120 125 Val Lys Ala Glu Gln Val Lys AlaSer Lys Glu Met Gly Glu Thr Leu 130 135 140 Leu Arg Ala Val Glu Ser TyrLeu Leu Ala His Ser Asp Ala Tyr Asn 145 150 155 160 3 160 PRT Betulaverrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 3 GlyVal Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys 20 25 30Val Ala Pro Gln Ala Ile Ser Ser Val Ser Asn Ile Glu Gly Asn Gly 35 40 45Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe 50 55 60Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Lys Phe Lys 65 70 7580 Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 9095 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asp His Glu Val115 120 125 Lys Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr LeuLeu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala TyrAsn Asn 145 150 155 160 4 160 PRT Betula verrucosa MISC_FEATURE(1)..(160) Point mutated Bet v 1 allergen 4 Gly Val Phe Asn Val Glu ThrGlu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala PheIle Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala IleSer Ser Val Glu Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile LysLys Ile Ser Phe Pro Glu Gly Phe Pro Phe 50 55 60 Lys Tyr Val Lys Asp ArgVal Asp Glu Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser ValIle Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn GluIle Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys IleSer Asn Lys Tyr His Thr Lys Gly Asp His Glu Val 115 120 125 Lys Ala GluGln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg AlaVal Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn 145 150 155 1605 160 PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 5 Gly Val Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly PhePro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Lys Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala HisSer Asp Ala Tyr Asn Asn 145 150 155 160 6 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 6 Gly Val Phe AsnVal Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu PheLys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala ProGln Ala Ile Ser Ser Val Glu Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro GlyThr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe 50 55 60 Lys Tyr ValLys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys 65 70 75 80 Tyr AsnTyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 Lys IleSer Asn Glu Ile Lys Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 IleLeu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asp His Glu Val 115 120 125Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn 145150 155 160 7 160 PRT Betula verrucosa MISC_FEATURE (1)..(160) Pointmutated Bet v 1 allergen 7 Gly Val Phe Asn Val Glu Thr Glu Thr Thr SerVal Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp GlyAsp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val GluAsn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser PhePro Glu Gly Phe Pro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val Asp Glu ValAsp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Glu Gly GlyPro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile ValAla Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys TyrHis Thr Lys Gly Asp His Glu Val 115 120 125 Lys Ala Glu Gln Val Glu AlaSer Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser TyrLeu Leu Ala His Ser Asp Ala Tyr Gly Asn 145 150 155 160 8 160 PRT Betulaverrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 8 GlyVal Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Lys 20 25 30Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser Gly Asn Gly 35 40 45Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe 50 55 60Lys Tyr Val Lys Asp Arg Val Asp Ser Val Asp His Thr Asn Phe Lys 65 70 7580 Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Ser 85 9095 Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Gly Asp Gly Gly Ser 100105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val115 120 125 Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu Thr LeuLeu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala TyrGly Asn 145 150 155 160 9 160 PRT Betula verrucosa MISC_FEATURE(1)..(160) Point mutated Bet v 1 allergen 9 Gly Val Phe Asn Val Glu ThrSer Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala PheIle Leu Thr Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala IleSer Ser Val Glu Asn Ile Ser Gly Lys Gly 35 40 45 Gly Pro Gly Thr Ile LysAsn Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys Tyr Val Lys Asp ArgVal Asp Glu Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser ValIle Ala Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn GluIle Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys IleSer Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val 115 120 125 Lys Ala GluGln Val Glu Ala Ser Lys Glu Met Gly Ser Thr Leu Leu 130 135 140 Arg AlaVal Glu Ser Tyr Leu Leu Ala His Thr Asp Ala Tyr Asn Asn 145 150 155 16010 160 PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v1 allergen 10 Gly Leu Phe Asn Val Glu Thr Ser Thr Thr Ser Val Ile ProAla Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Thr Gly Asp Thr LeuPhe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile SerGly Lys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Asn Ile Ser Phe Pro Glu GlyLeu Pro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val Asp Thr Val Asp His ThrLys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Ala Gly Gly Pro Ile GlyAsp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr GlyAsp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr LysGly Tyr His Glu Val 115 120 125 Lys Val Glu Gln Val Glu Ala Ser Lys GluMet Gly Ser Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu AlaHis Thr Asp Ala Tyr Asn Asn 145 150 155 160 11 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 11 Gly Leu PheAsn Val Glu Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Lys Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Thr Val Asp His Thr Thr Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asn Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Lys Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn145 150 155 160 12 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 12 Gly Leu Phe Asn Val Ser Thr Ser ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Ser Asn Ile Ser Gly Lys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspThr Val Asp His Thr Thr Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asn Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Lys Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130 135 140 Arg Ala Val GluSer Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 13 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 13 Gly Val Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser GlyLys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Asn Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Lys Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Asn Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala HisThr Asp Ala Tyr Asn Asn 145 150 155 160 14 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 14 Gly Leu PheAsn Val Glu Thr Ser Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser Gly Lys Gly 35 40 45 Gly ProGly Thr Ile Lys Asn Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys TyrVal Lys Asp Arg Val Asp Thr Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val 115 120125 Lys Val Glu Gln Val Glu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Thr Asp Ala Tyr Asn Asn145 150 155 160 15 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 15 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspGlu Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Lys Gly Tyr His Glu Val 115 120 125 Lys Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130 135 140 Arg Ala Val GluSer Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 16 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 16 Gly Leu Phe Asn Val Glu Thr Ser Thr Pro Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser GlyLys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Glu Val Asp Thr Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Lys Val Glu Gln Val Glu Ala Ser Lys Glu MetGly Asn Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 17 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 17 Gly Val PheAsn Val Glu Thr Ser Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser Gly Lys Gly 35 40 45 Gly ProGly Thr Ile Lys Asn Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys TyrVal Lys Asp Arg Val Asp Ser Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 LysIle Ser Asn Glu Ile Lys Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Asn Tyr His Thr Lys Gly Tyr His Glu Val 115 120125 Lys Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Gly Asn145 150 155 160 18 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 18 Gly Leu Phe Asn Val Ser Thr Ser ThrThr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Glu Asn Ile Ser Gly Lys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Asn IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val AspSer Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 Lys Ile Ser Asn Glu Ile ValIle Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnAsn Tyr His Thr Lys Gly Tyr His Glu Val 115 120 125 Lys Val Glu Gln ValGlu Ala Ser Lys Glu Met Gly Asn Thr Leu Leu 130 135 140 Arg Ala Val GluSer Tyr Leu Leu Ala His Thr Asp Ala Tyr Gly Asn 145 150 155 160 19 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 19 Gly Val Phe Asn Val Glu Thr Glu Thr Pro Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Ser Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly AspThr Leu Ser 85 90 95 Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Lys Ala Glu Gln Val Lys Ala Ser Lys Asn MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala HisSer His Ala Tyr Gly Asn 145 150 155 160 20 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 20 Gly Leu PheAsn Val Ser Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Ser Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 LysIle Ser Asn Glu Ile Val Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val 115 120125 Lys Val Glu Gln Val Lys Ala Ser Lys Asn Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Thr His Ala Tyr Gly Asn145 150 155 160 21 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 21 Gly Val Phe Asn Val Glu Thr Ser ThrThr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Glu Asn Ile Ser Gly Lys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Asn IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val AspGlu Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 Lys Ile Ser Asn Glu Ile LysIle Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnAsn Tyr His Thr Lys Gly Asp His Glu Val 115 120 125 Lys Ala Glu Gln ValGlu Ala Ser Lys Asn Met Gly Asn Thr Leu Leu 130 135 140 Arg Ala Val GluSer Tyr Leu Leu Ala His Thr Asp Ala Tyr Asn Asn 145 150 155 160 22 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 22 Gly Leu Phe Asn Val Ser Thr Ser Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser GlyLys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Asn Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Lys Tyr Val Lys Asp Asn Val Asp Glu Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Ala Gly Gly Pro Ile Gly AspThr Leu Ser 85 90 95 Lys Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Asn Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Lys Val Glu Gln Val Glu Ala Ser Lys Asn MetGly Asn Thr Leu Leu 130 135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala HisThr Asp Ala Tyr Asn Asn 145 150 155 160 23 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 23 Gly Val PheAsn Val Glu Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Glu Val Asp His Ala Asn Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 LysIle Ser Asn Glu Ile Val Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val 115 120125 Lys Ala Glu Gln Val Lys Ala Ser Lys Asn Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn145 150 155 160 24 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 24 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspGlu Val Asp His Ala Asn Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Asp Thr Leu Ser 85 90 95 Lys Ile Ser Asn Glu Ile ValIle Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnAsn Tyr His Thr Lys Gly Tyr His Glu Val 115 120 125 Lys Val Glu Gln ValLys Ala Ser Lys Asn Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluSer Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 25 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 25 Gly Val Phe Asn Val Glu Thr Glu Thr Ala Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Leu Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Pro Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile GlyAsp His Glu Val 115 120 125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Gly Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 26 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 26 Gly Val PheAsn Val Glu Thr Glu Thr Ala Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Leu Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Pro Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Gly Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn145 150 155 160 27 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 27 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe His Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Lys Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspArg Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile GluGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Glu Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluGly Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 28 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 28 Gly Val Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe His Ala Phe Ile Leu Asp Gly Asp Asn Leu PhePro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Ser Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile GlyAsp His Glu Val 115 120 125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Arg Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 29 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 29 Gly Val PheAsn Val Glu Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Met Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Ser Val Asp His Thr Asn Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Ala Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn145 150 155 160 30 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 30 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Leu Ala Phe Ile LeuAsp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspThr Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile GlyGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Glu Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluArg Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 31 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 31 Gly Val Phe Asn Val Glu Thr Glu Thr Pro Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Gln Ala Phe Ile Leu Asp Gly Asp Asn Leu PhePro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Ser Val Asp His Thr AsnPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile GlyAsp His Glu Val 115 120 125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Pro Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 32 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 32 Gly Val PheAsn Val Glu Thr Glu Thr Ala Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Leu Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val AlaPro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Gly Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Asp Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Arg Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn145 150 155 160 33 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 33 Gly Val Phe Asn Val Glu Thr Glu ThrAla Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Leu Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Glu Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspGly Val Asp His Thr Asn Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile GlyGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Glu Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluAla Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 34 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 34 Gly Val Phe Asn Val Glu Thr Glu Thr Pro Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu PhePro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Ser Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile GlyAsp His Glu Val 115 120 125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Thr Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 35 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 35 Gly Val PheAsn Val Glu Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val AlaPro Pro Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Arg Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Arg Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn145 150 155 160 36 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 36 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspArg Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile GlyGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Glu Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluArg Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 37 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 37 Gly Val Phe Asn Val Glu Thr Glu Thr Pro Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Leu Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Lys Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Arg Val Asp His Thr LysPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Asp Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Pro AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile GlyAsp His Glu Val 115 120 125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Pro Tyr Leu Leu Ala HisSer His Ala Tyr Asn Asn 145 150 155 160 38 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 38 Gly Val PheAsn Val Glu Thr Glu Thr Pro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Leu Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Lys Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Gly Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Gly Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Ile Gly Asp His Glu Val 115 120125 Glu Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Thr Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn145 150 155 160 39 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 39 Gly Val Phe Asn Val Glu Thr Glu ThrPro Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Met Ala Phe Ile LeuAsp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val Ala Pro Pro Ala Ile Ser SerVal Ser Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspGly Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AspGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Ile Gly Asp His Glu Val 115 120 125 Glu Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluGly Tyr Leu Leu Ala His Ser His Ala Tyr Asn Asn 145 150 155 160 40 160PRT Betula verrucosa MISC_FEATURE (1)..(160) Point mutated Bet v 1allergen 40 Gly Val Phe Asn Val Glu Thr Glu Thr Asn Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Ser Gly LeuPro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr AsnPhe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly AspThr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly AspGly Gly Ser 100 105 110 Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys GlyTyr His Glu Val 115 120 125 Asn Ala Glu Gln Val Lys Ala Ser Lys Glu MetGly Glu Thr Leu Leu 130 135 140 Arg Ala Val Glu Thr Tyr Leu Leu Ala HisSer Asp Ala Tyr Asn Asn 145 150 155 160 41 160 PRT Betula verrucosaMISC_FEATURE (1)..(160) Point mutated Bet v 1 allergen 41 Gly Leu PheAsn Val Glu Thr Glu Thr Asn Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Ser Asn Ile Ser Gly Lys Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Ser Gly Leu Pro Phe 50 55 60 Asn TyrVal Lys Asp Arg Val Asp Glu Val Asp His Asn Asn Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 SerIle Ser Asn Glu Ile Val Ile Val Ala Thr Gly Asp Gly Gly Ser 100 105 110Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu Val 115 120125 Asn Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130135 140 Arg Ala Val Glu Thr Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn145 150 155 160 42 160 PRT Betula verrucosa MISC_FEATURE (1)..(160)Point mutated Bet v 1 allergen 42 Gly Leu Phe Asn Val Glu Thr Glu ThrAsn Ser Val Ile Pro Ala Gly 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Asn Ala Ile Ser SerVal Ser Asn Ile Ser Gly Lys Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Ser Gly Leu Pro Phe 50 55 60 Asn Tyr Val Lys Asp Arg Val AspSer Val Asp His Asn Asn Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile AlaGly Gly Pro Ile Gly Ser Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Gly Asp Gly Gly Ser 100 105 110 Ile Leu Lys Ile Ser AsnLys Tyr His Thr Lys Gly Tyr His Glu Val 115 120 125 Asn Ala Glu Gln ValGlu Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135 140 Arg Ala Val GluThr Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn 145 150 155 160 43 161PRT Betula verrucosa MISC_FEATURE (1)..(161) Point mutated Bet v 1allergen 43 Gly Val Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Xaa GlyPhe Pro 50 55 60 Phe Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His ThrLys Phe 65 70 75 80 Lys Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile GlyAsp Thr Leu 85 90 95 Glu Ser Ile Ser Asn Glu Ile Val Ile Val Ala Thr GlyAsp Gly Gly 100 105 110 Ser Ile Leu Lys Ile Ser Asn Lys Tyr His Thr LysGly Tyr His Glu 115 120 125 Val Lys Ala Glu Gln Val Glu Ala Ser Lys GluMet Gly Glu Thr Leu 130 135 140 Leu Arg Ala Val Glu Ser Tyr Leu Leu AlaHis Ser Asp Ala Tyr Asn 145 150 155 160 Asn 44 161 PRT Betula verrucosaMISC_FEATURE (1)..(161) Point mutated Bet v 1 allergen 44 Gly Val PheAsn Val Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg LeuPhe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val AlaPro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser Gly Asn Gly 35 40 45 Gly ProGly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe 50 55 60 Lys TyrVal Lys Asp Arg Val Asp Glu Val Asp His Thr Lys Phe Lys 65 70 75 80 TyrAsn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Xaa Thr Leu 85 90 95 GluSer Ile Ser Asn Glu Ile Val Ile Val Ala Thr Gly Asp Gly Gly 100 105 110Ser Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Tyr His Glu 115 120125 Val Lys Ala Glu Gln Val Glu Ala Ser Lys Glu Met Gly Glu Thr Leu 130135 140 Leu Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn145 150 155 160 Asn 45 161 PRT Betula verrucosa MISC_FEATURE (1)..(161)Point mutated Bet v 1 allergen 45 Gly Val Phe Asn Val Glu Thr Glu ThrThr Ser Val Xaa Ile Pro Ala 1 5 10 15 Ala Arg Leu Phe Lys Ala Phe IleLeu Asp Gly Asp Thr Leu Phe Pro 20 25 30 Gln Val Ala Pro Gln Ala Ile SerSer Val Glu Asn Ile Ser Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys LysIle Ser Phe Pro Glu Gly Phe Pro 50 55 60 Phe Lys Tyr Val Lys Asp Arg ValAsp Glu Val Asp His Thr Lys Phe 65 70 75 80 Lys Tyr Asn Tyr Ser Val IleGlu Gly Gly Pro Ile Gly Asp Thr Leu 85 90 95 Glu Ser Ile Ser Asn Glu IleVal Ile Val Ala Thr Gly Asp Gly Gly 100 105 110 Ser Ile Leu Lys Ile SerAsn Lys Tyr His Thr Lys Gly Tyr His Glu 115 120 125 Val Lys Ala Glu GlnVal Glu Ala Ser Lys Glu Met Gly Glu Thr Leu 130 135 140 Leu Arg Ala ValGlu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn 145 150 155 160 Asn 46162 PRT Betula verrucosa MISC_FEATURE (1)..(162) Point mutated Bet v 1allergen 46 Gly Val Phe Asn Val Glu Thr Glu Thr Thr Ser Val Ile Pro AlaAla 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Thr Leu PhePro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Ser GlyAsn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys Ile Xaa Ser Phe Pro Glu XaaGly Phe 50 55 60 Pro Phe Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp HisThr Lys 65 70 75 80 Phe Lys Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro IleGly Asp Thr 85 90 95 Leu Glu Ser Ile Ser Asn Glu Ile Val Ile Val Ala ThrGly Asp Gly 100 105 110 Gly Ser Ile Leu Lys Ile Ser Asn Lys Tyr His ThrLys Gly Tyr His 115 120 125 Glu Val Lys Ala Glu Gln Val Glu Ala Ser LysGlu Met Gly Glu Thr 130 135 140 Leu Leu Arg Ala Val Glu Ser Tyr Leu LeuAla His Ser Asp Ala Tyr 145 150 155 160 Asn Asn 47 159 PRT Betulaverrucosa MISC_FEATURE (1)..(159) Point mutated Bet v 1 allergen (aminoacid deletion of residue G111) 47 Gly Val Phe Asn Val Glu Thr Glu ThrThr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu Phe Lys Ala Phe Ile LeuAsp Gly Asp Thr Leu Phe Pro Gln 20 25 30 Val Ala Pro Gln Ala Ile Ser SerVal Glu Asn Ile Ser Gly Asn Gly 35 40 45 Gly Pro Gly Thr Ile Lys Lys IleSer Phe Pro Glu Gly Phe Pro Phe 50 55 60 Lys Tyr Val Lys Asp Arg Val AspGlu Val Asp His Thr Lys Phe Lys 65 70 75 80 Tyr Asn Tyr Ser Val Ile GluGly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Ser Ile Ser Asn Glu Ile ValIle Val Ala Thr Gly Asp Gly Ser Ile 100 105 110 Leu Lys Ile Ser Asn LysTyr His Thr Lys Gly Tyr His Glu Val Lys 115 120 125 Ala Glu Gln Val GluAla Ser Lys Glu Met Gly Glu Thr Leu Leu Arg 130 135 140 Ala Val Glu SerTyr Leu Leu Ala His Ser Asp Ala Tyr Asn Asn 145 150 155 48 24 DNA Betulaverrucosa 48 cagactaatt cgagctcggt accc 24 49 24 DNA Betula verrucosa 49gaaggctttc ctttcaaata cgtg 24 50 24 DNA Betula verrucosa 50 tttgaaagggaggccttcgg gaaa 24 51 24 DNA Betula verrucosa 51 gacacattgc tgaagatctccaac 24 52 24 DNA Betual verrucosa 52 ggagatcttc agcaatgtgt cgcc 24 5324 DNA Betula verrucosa 53 gtggcaaccc cggatggagg atcc 24 54 24 DNABetula verrucosa 54 tcctccatcc ggggttgcca ctat 24 55 24 DNA Betulaverrucosa 55 accaaaggtt atcatgaggt gaag 24 56 24 DNA Betula verrucosa 56cacctcatga taacctttgg tgtg 24 57 38 DNA Betula verrucosa 57 gccggaattcattagttgtt gtaggcatcg gagtgtgc 38 58 24 DNA Betula verrucosa primer 58actgagaccv cstctgttat ccca 24 59 24 DNA Betula verrucosa 59 gataacagasgbggtctcag tctc 24 60 24 DNA Betula verrucosa 60 cgactgttcm wkgcctttatcctt 24 61 24 DNA Betula verrucosa 61 gataaaggcm wkgaacagtc gagc 24 6224 DNA Betula verrucosa 62 gttgcacccv mrgccattag cagt 24 63 24 DNABetula verrucosa 63 gctaatggcy kbgggtgcaa cctt 24 64 24 DNA Betulaverrucosa 64 agcgtgatcg rwggcggtcc cata 24 65 24 DNA Betula verrucosa 65gggaccgccw ycgatcacgc tgta 24 66 27 DNA Betula verrucosa 66 attggtgaccatgaggtgra wgcagag 27 67 27 DNA Betula verrucosa 67 wtycacctcatggtcaccaa tggtgtg 27 68 24 DNA Betula verrucosa 68 gccgttgagvsytacctctt ggca 24 69 24 DNA Betula verrucosa 69 caagaggtar sbctcaacggccct 24 70 24 DNA Betula verrucosa 70 agagttgatv sygtggacca caca 24 7124 DNA Betula verrucosa 71 gtggtccacr sbatcaactc tgtc 24 72 30 DNABetula verrucosa 72 cgtcccaagc tttcattagt tgttgtaggc 30 73 38 DNA Betulaverrucosa 73 cggaattcat aatggattat aaagatggtg tgtttaat 38 74 24 DNABetula verrucosa 74 cagactaatt cgagctcggt accc 24 75 23 DNA Betulaverrucosa 75 tttcctgaaa tgttttcaac act 23 76 23 DNA Betula verrucosa 76aacatttcag gaaatggagg gcc 23 77 24 DNA Betula verrucosa 77 cacgtagttgaaagggaggc cttc 24 78 24 DNA Betula verrucosa 78 tttcaactac gtgaaggacagagt 24 79 24 DNA Betula verrucosa 79 tttcaactac gtgaaggaca gagt 24 8024 DNA Betula verrucosa 80 tttcaactac gtgaaggaca gagt 24 81 24 DNABetula verrucosa 81 ggagagcatc tccaacgaga taaa 24 82 24 DNA Betulaverrucosa 82 caggttgaag caagtaaaga aatg 24 83 24 DNA Betula verrucosa 83gcaggtcgac tctagaggat ccat 24 84 24 DNA Betula verrucosa 84 cagactaattcgacgtcggt accc 24 85 24 DNA Betula verrucosa 85 cagtcgcggt gctgggataacaga 24 86 24 DNA Betula verrucosa 86 ccagcaccgc gactgttcaa ggcc 24 8724 DNA Betula verrucosa 87 cactatggtt atctcgttgg agat 24 88 24 DNABetula verrucosa 88 gagataacca tagtggcaac tggt 24 89 45 DNA Betulaverrucosa 89 ttactgaatt cattagttgt aggcatccgg gtggcctttg aggta 45

1. A recombinant Bet v 1 allergen, characterised in that it is a mutantof a naturally occurring Bet v 1 allergen wherein: a. the mutant retainsessentially the same α-carbon backbone structure as the naturallyoccurring allergen, b. the mutant comprises at least four primarymutations, which each reduce the specific IgE binding capability of themutated allergen as compared to the IgE binding capability of thenaturally occurring Bet v 1 allergen, c. each primary mutation is asubstitution of one surface-exposed amino acid residue with anotherresidue, d. the mutations are placed in such a manner that at least onearea of 400-800 Å² comprises either no mutations or one or more moderatemutations, e. the primary mutations are selected from at least 4 of thefollowing 10 groups, each group comprising surface exposed amino acidpositions suitable for amino acid substitution: group 1: A130, E131,K134, A135, K137, E138, E141, T142, R145; group 2: V2, F3, N4, Y5, E6,T7, K119; group 3: D27, S39, S40, Y41, E42, N43, I44, E45, G46, N47,P50, G51, K55, D72, E73; group 4: E8, T10, V12, P14, V105, A106, T107,P108, D109, G 10, I113, K115; group 5: A16, K20, S149, Y150, L152, A153,H154, S155, D156, Y158, N159, +160, wherein +160 represents addition ofan N-terminal amino acid; group 6: L24, D25, N28, K32; group 7: H76,T77, N78, F79, K80, E101, K103; group 8: K68, R70, I86, E87, E96, K97;group 9: G1, G92, D93, T94, K123, G124, D125, H126, E127, K129; group10: P35, Q36, E60, G61, P63, F64, K65, Y66; with the proviso that therecombinant Bet v 1 allergen is not one of the following specificmutants: (Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu, Asp156His,+160Asn); (Tyr5Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123Ile,Lys134Glu, Asp156His); (Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser,Lys134Glu); (Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly,Leu152Lys, Ala153Gly, Ser155Pro); (N28T, K32Q, N78K, K103V, P108G,R145E, D156H, +160N); (Tyr5Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu45Ser,Lys65Asn, Asn78Lys, Lys103Val, Lys97Ser, Pro108Gly, Arg145Glu,Asp156His, +160Asn); (Tyr5Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys,Lys103Val, Lys123Ile, Asp156His, +160Asn); (Asn28Thr, Lys32Gln,Lys65Asn, Glu96Leu, Lys97Ser, Pro108Gly, Asp109Asn, Asp125Tyr,Glu127Ser, Arg145Glu); (Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G,K123I, K134E, D156H, +160N); (Y5V, E42S, E45S, K65N, N78K, K97S, K103V,K123I, K134E, D156H, +160N); and (Y5V, N28T, K32Q, E42S, E45S, K65N,N78K, K97S, K103V, P108G, K123I, K134E, D156H, +160N).
 2. A recombinantBet v 1 allergen according to claim 1, wherein the primary mutations areselected from at least 4 of the following 10 groups, each groupcomprising surface exposed amino acid positions suitable for amino acidsubstitution: group 1: A130, K134, A135, K137, E138, E141, T142, R145;group 2: V2, F3, N4, Y5, E6, T7, K119; group 3: D27, Y41, E42, N43, I44,E45, G46, N47, P50, G51, K55, D72, E73; group 4: E8, T10, P108, D109,I113, K115; group 5: H154, S155, D156, N159, +160; group 6: D25, N28,K32; group 7: H76, T77, N78, K80, E101, K103; group 8: K68, R70, I86,E87, E96, K97; group 9: G1, G92, T94, K123, G124, D125, H126; group 10:K65, Y66.
 3. A recombinant Bet v 1 allergen according to claim 2,wherein the primary mutations are selected from at least 4 of thefollowing 10 groups, each group comprising surface exposed amino acidpositions suitable for amino acid substitution: group 1: A130, K134,A135, K137, E138, E141, T142; group 2: V2, F3, N4, Y5, E6, T7, K119;group 3: D27, Y41, N43, I44, E45, G46, N47, P50, G51, K55, D72, E73;group 4: E8, P108, I113, K115; group 5: H154, S155, N159, +160; group 6:D25, N28; group 7: H76, N78, K80, E101, K103; group 8: K68, R70, I86,E87, E96, K97; group 9: G1, G92, T94, G124, D125, H126; group 10: Y66.4. A recombinant Bet v 1 allergen according to claim 1, wherein theprimary mutations are selected from at least 4 of the following 9groups, each group comprising surface exposed amino acid positionssuitable for amino acid substitution: group 1: A130, A135, K137, E138,E141, T142; group 2: F3, N4, E6, T7, K119; group 3: D27, Y41, N43, I44,G46, P50, G51, D72, E73; group 4: E8, I113, K115; group 5: H154, S155,N159; group 7: H76, N78, K80, E101; group 8: K68, R70, I86, E87; group9: G1, G92, D93, G124, H126; group 10: Y66.
 5. A recombinant Bet v 1allergen according to claim 1, where the primary mutations are selectedfrom at least 4 of the following 10 groups, each group comprisingsurface exposed amino acid positions suitable for the following specificamino acid substitutions: group 1: A130: A130V, A130G, A130I, A130L,A130S, A130H, A130T; E131: E131D, E131H, E131K, E131R, E131S; K134:K134R, K134H, K134S, K134Q, K1341, K134E; A135: A135V, A135G, A135I,A135L, A135S, A135H, A135T; K137: K137R, K137H, K137S, K137Q, K137I,K137E; E138: E138D, E138H, E138K, E138R, E138S, E138N; E141: E141D,E141H, E141K, E141R, E141S; T142: T142A, T142S, T142L, T142V, T142D,T142K, T142N; R145: R145K, R145H, R145T, R145D, R145E; group 2: V2: V2A,V2I, V2K, V2L, V2R, V2T; F3: F3H, F3W, F3S, F3D; N4: N4H, N4K, N4M, N4Q,N4R; Y5: Y5D, Y5G, Y5H, Y5I, Y5K, Y5V; E6: E6D, E6H, E6K, E6R, E6S; T7:T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119: K119R, K119H, K119S, K119Q,K119I, K119E, K119N; group 3: D27: D27E, D27H, D27K, D27R, D27S; S39:S39T, S39L, S39V, S39D, S39K; S40: S40T, S40L, S40V, S40D, S40K; Y41:Y41D, Y41G, Y41H, Y41I, Y41K, Y41V; E42: E42S, E42D, E42H, E42K, E42R;N43: N43H, N43K, N43M, N43Q, N43R; I44:I44L, I44K, I44R, I44D; E45:E45S, E45D, E45H, E45K, E45R; G46: G46N, G46H, G46K, G46M, G46Q, G46R;N47: N47H, N47K, N47M, N47Q, N47R; P50: P50G; G51: G51N, G51H, G51K,G51M, G51Q, G51R; K55: K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72:D72E, D72S, D72H, D72R, D72K; E73: E73D, E73S, E73H, E73R, E73K; group4: E8: E8D, E8H, E8K, E8R, E8S; T10: T10P, T10S, T10L, T10V, T10D, T10K,T10N; V12: V12A, V12I, V12K, V12L, V12R, V12T; P14: P14G; V105: V105A,V105I, V105K, V105L, V105R, V105T; A106: A106V, A106G, A106I, A106L,A106S, A106H, A106T; T107: T107A, T107S, T107L, T107V, T107D, T107K,T107N; P108: P108G; D109: D109N D109E, D109S, D109H, D109R, D109K; G110:G110N, G110H, G110K, G110M, G110Q, G110R; I113: I113L, I113K, I113R,I113D, K115: K115R, K115H, K115S, K115Q, K115I, K115E, K115N; group 5:A16: A16V, A16G, A16I, A16L, A16S, A16H, A16T; K20: K20R, K20H, K20S,K20Q, K20I, K20E, K20N; S149: S149T, S149L, S149V, S149D, S149K; Y150:Y150T, Y150L, Y150V, Y150D, Y150K; L152: L152A, L152V, L152G, L152I,L152S, L152H, L152T; A153: A153V, A153G, A1531, A153L, A153S, A153H,A153T; H154: H154W, H154F, H154S, H154D; S155: S155T, S155L, S155V,S155D, S155K; D156: D156H, D156E, D156S, D156R, D156K; Y158: Y158D,Y158G, Y158H, Y158I, Y158K, Y158V; N159: N159H, N159K, N159M, N159Q,N159R, N159G, +160N; group 6: L24: L24A, L24V, L24G, L24I, L24S, L24H,L24T; D25: D25E, D25H, D25K, D25R, D25S; N28: N28H, N28K, N28M, N28Q,N28R, N28T; K32: K32Q, K32R, K32N, K32H, K32S, K32I, K32E; group 7: H76:H76W, H76F, H76S, H76D; T77: T77A, T77S, T77L, T77V, T77D, T77K, T77N;N78: N78H, N78K, N78M, N78Q, N78R; F79: F79H, F79W, F79S, F79D; K80:K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101: E101D, E101H, E101K,E101R, E101S; K103: K103R, K103H, K103S, K103Q, K103I, K103E, K103V;group 8: K68: K68R, K68H, K68S, K68Q, K68I, K68E, K68N; R70: R70K, R70H,R70T, R70D, R70E, R70N; I86: I86L, I86K, I86R, I86D; E87: E87D, E87H,E87K, E87R, E87S, E87A; E96: E96D, E96H, E96K, E96R, E96S, E96L; K97:K97R, K97H, K97S, K97Q, K97I, K97E; group 9: G1: G1N, G1H, G1K, G1M,G1Q, G1R; G92: G92N, G92H, G92K, G92M, G92Q, G92R; D93: D93N, D93E,D93S, D93H, D93R, D93K; T94: T94A, T94S, T94L, T94V, T94D, T94K, T94N;K123: K123R, K123H, K123S, K123Q, K123I, K123E; G124: G124N, G124H,G124K, G124M, G124Q, G124R; D125: D125E, D125H, D125K, D125R, D125S,D125Y; H126: H126W, H126F, H126S, H126D; E127: E127D, E127H, E127K,E127R, E127S; K129: K129R, K129H, K129S, K129Q, K129I, K129E, K129N;group 10: P35: P35G; Q36: Q36K, Q36R, Q36N, Q36H, Q36S, Q36I, Q36E; E60:E60H, E60K, E60M, E60Q, E60R; G61: G61N, G61H, G61K, G61M, G61Q, G61R;P63: P63G; F64: F64H, F64W, F64S, F64D; K65: K65R, K65H, K65S, K65Q,K651, K65E, K65N; Y66: Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.
 6. Arecombinant Bet v 1 allergen according to claim 1, where the primarymutations are selected from at least 4 of the following 10 groups, eachgroup comprising surface exposed amino acid positions suitable for thefollowing specific amino acid substitutions: group 1: A130: A130V,A130G, A130I, A130L, A130S, A130H, A130T; K134: K134R, K134H, K134S,K134Q, K134I, K134E; A135: A135V, A135G, A135I, A135L, A135S, A135H,A135T; K137: K137R, K137H, K137S, K137Q, K137I, K137E; E138: E138D,E138H, E138K, E138R, E138S, E138N; E141: E141D, E141H, E141K, E141R,E141S; T142: T142A, T142S, T142L, T142V, T142D, T142K, T142N; R145:R145K, R145H, R145T, R145D, R145E; group 2: V2: V2A, V2I, V2K, V2L, V2R,V2T; F3: F3H, F3W, F3S, F3D; N4: N4H, N4K, N4M, N4Q, N4R; Y5: Y5D, Y5G,Y5H, Y5I, Y5K, Y5V; E6: E6D, E6H, E6K, E6R, E6S; T7: T7P, T7S, T7L, T7V,T7D, T7K, T7N; K119: K119R, K119H, K119S, K119Q, K119I, K119E, K119N;group 3: D27: D27E, D27H, D27K, D27R, D27S; Y41: Y41D, Y41G, Y41H, Y41I,Y41K, Y41V; E42: E42S, E42D, E42H, E42K, E42R; N43: N43H, N43K, N43M,N43Q, N43R; I44: I44L, I44K, I44R, I44D; E45: E45S, E45D, E45H, E45K,E45R; G46: G46N, G46H, G46K, G46M, G46Q, G46R; N47: N47H, N47K, N47M,N47Q, N47R; P50: P50G; G51: G51N, G51H, G51K, G51M, G51Q, G51R; K55:K55R, K55H, K55S, K55Q, K55I, K55E, K55N; D72: D72E, D72S, D72H, D72R,D72K; E73: E73D, E73S, E73H, E73R, E73K; group 4: E8: E8D, E8H, E8K,E8R, E8S; T10: T10P, T10S, T10L, T10V, T10D, T10K, T10N; P108: P108G;D109: D109N D109E, D109S, D109H, D109R, D109K; I113: I113L, I113K,I113R, I113D, K115: K115R, K115H, K115S, K115Q, K115I, K115E, K115N;group 5: H154: H154W, H154F, H154S, H154D; S155: S155T, S155L, S155V,S155D, S155K; D156: D156H, D156E, D156S, D156R, D156K; N159: N159H,N159K, N159M, N159Q, N159R, N159G, +160N; group 6: D25: D25E, D25H,D25K, D25R, D25S; N28: N28H, N28K, N28M, N28Q, N28R, N28T; K32: K32Q,K32R, K32N, K32H, K32S, K32I, K32E; group 7: H76: H76W, H76F, H76S,H76D; T77: T77A, T77S, T77L, T77V, T77D, T77K, T77N; N78: N78H, N78K,N78M, N78Q, N78R; K80: K80R, K80H, K80S, K80Q, K80I, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; K103: K103R, K103H, K103S, K103Q,K103I, K103E, K103V; group 8: K68: K68R, K68H, K68S, K68Q, K68I, K68E,K68N; R70: R70K, R70H, R70T, R70D, R70E, R70N; I86: I86L, I86K, I86R,I86D; E87: E87D, E87H, E87K, E87R, E87S, E87A; E96: E96D, E96H, E96K,E96R, E96S, E96L; K97: K97R, K97H, K97S, K97Q, K971, K97E; group 9: G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92: G92N, G92H, G92K, G92M, G92Q, G92R;T94: T94A, T94S, T94L, T94V, T94D, T94K, T94N; K123: K123R, K123H,K123S, K123Q, K123I, K123E; G124: G124N, G124H, G124K, G124M, G124Q,G124R; D125: D125E, D125H, D125K, D125R, D125S, D125Y; H126: H126W,H126F, H126S, H126D; group 10: K65: K65R, K65H, K65S, K65Q, K65I, K65E,K65N; Y66: Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.
 7. A recombinant Bet v 1allergen according to claim 1, where the primary mutations are selectedfrom at least 4 of the following 10 groups, each group comprisingsurface exposed amino acid positions suitable for the following specificamino acid substitutions: group 1: A130: A130V, A130G, A130I, A130L,A130S, A130H, A130T; K134: K134R, K134H, K134S, K134Q, K134I, K134E;A135: A135V, A135G, A135I, A135L, A135S, A135H, A135T; K137: K137R,K137H, K137S, K137Q, K137I, K137E; E138: E138D, E138H, E138K, E138R,E138S, E138N; E141: E141D, E141H, E141K, E141R, E141S; T142: T142A,T142S, T142L, T142V, T142D, T142K, T142N; group 2: V2: V2A, V2I, V2K,V2L, V2R, V2T; F3: F3H, F3W, F3S, F3D; N4: N4H, N4K, N4M, N4Q, N4R; Y5:Y5D, Y5G, Y5H, Y5I, Y5K, Y5V; E6: E6D, E6H, E6K, E6R, E6S; T7: T7P, T7S,T7L, T7V, T7D, T7K, T7N; K119: K119R, K119H, K119S, K119Q, K119I, K119E,K119N; group 3: D27: D27E, D27H, D27K, D27R, D27S; Y41: Y41D, Y41G,Y41H, Y41I, Y41K, Y41V; N43: N43H, N43K, N43M, N43Q, N43R; I44: I44L,I44K, I44R, I44D; E45: E45S, E45D, E45H, E45K, E45R; G46: G46N, G46H,G46K, G46M, G46Q, G46R; N47: N47H, N47K, N47M, N47Q, N47R; P50: P50G;G51: G51N, G51H, G51K, G51M, G51Q, G51R; K55: K55R, K55H, K55S, K55Q,K551, K55E, K55N; D72: D72E, D72S, D72H, D72R, D72K; E73: E73D, E73S,E73H, E73R, E73K; group 4: E8: E8D, E8H, E8K, E8R, E8S; P108: P108G;I113: I113L, I113K, I113R, I113D, K 15: K115R, K115H, K115S, K115Q,K115I, K115E, K115N; group 5: H154: H154W, H154F, H154S, H154D; S155:S155T, S155L, S155V, S155D, S155K; N159: N159H, N159K, N159M, N159Q,N159R, N159G, +160N; group 6: D25: D25E, D25H, D25K, D25R, D25S; N28:N28H, N28K, N28M, N28Q, N28R, N28T; group 7: H76: H76W, H76F, H76S,H76D; N78: N78H, N78K, N78M, N78Q, N78R; K80: K80R, K80H, K80S, K80Q,K80I, K80E, K80N; E101: E101D, E101H, E101K, E101R, E101S; K103: K103R,K103H, K103S, K103Q, K103I, K103E, K103V; group 8: K68: K68R, K68H,K68S, K68Q, K68I, K68E, K68N; R70: R70K, R70H, R70T, R70D, R70E, R70N;I86: I86L, I86K, I86R, I86D; E87: E87D, E87H, E87K, E87R, E87S, E87A;E96: E96D, E96H, E96K, E96R, E96S, E96L; K97: K97R, K97H, K97S, K97Q,K971, K97E; group 9: G1: G1N, G1H, G1K, G1M, G1Q, G1R; G92: G92N, G92H,G92K, G92M, G92Q, G92R; T94: T94A, T94S, T94L, T94V, T94D, T94K, T94N;G124: G124N, G124H, G124K, G124M, G124Q, G124R; D125: D125E, D125H,D125K, D125R, D125S, D125Y; H126: H126W, H126F, H126S, H126D; group 10:Y66: Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.
 8. A recombinant Bet v 1allergen according to claim 1, where the primary mutations are selectedfrom at least 4 of the following 8 groups, each group comprising surfaceexposed amino acid positions suitable for the following specific aminoacid substitutions: group 1: A130: A130V, A130G, A130I, A130L, A130S,A130H, A130T; A135: A135V, A135G, A135I, A135L, A135S, A135H, A135T;K137: K137R, K137H, K137S, K137Q, K137I, K137E; E138: E138D, E138H,E138K, E138R, E138S, E138N; E141: E141D, E141H, E141K, E141R, E141S;T142: T142A, T142S, T142L, T142V, T142D, T142K, T142N; group 2: F3: F3H,F3W, F3S, F3D; N4: N4H, N4K, N4M, N4Q, N4R; E6: E6D, E6H, E6K, E6R, E6S;T7: T7P, T7S, T7L, T7V, T7D, T7K, T7N; K119: K119R, K119H, K119S, K119Q,K119I, K119E, K119N; group 3: D27: D27E, D27H, D27K, D27R, D27S; Y41:Y41D, Y41G, Y41H, Y41I, Y41K, Y41V; N43: N43H, N43K, N43M, N43Q, N43R;I44: I44L, I44K, I44R, I44D; G46: G46N, G46H, G46K, G46M, G46Q, G46R;N47: N47H, N47K, N47M, N47Q, N47R; P50: P50G; G51: G51N, G51H, G51K,G51M, G51Q, G51R; D72: D72E, D72S, D72H, D72R, D72K; E73: E73D, E73S,E73H, E73R, E73K; group 4: E8: E8D, E8H, E8K, E8R, E8S; I113: I113L,I113K, I113R, I113D, K115: K115R, K115H, K115S, K115Q, K115I, K115E,K115N; group 5: H154: H154W, H154F, H154S, H154D; S155: S155T, S155L,S155V, S155D, S155K; N159: N159H, N159K, N159M, N159Q, N159R,N159G,+160N; group 7: H76: H76W, H76F, H76S, H76D; N78: N78H, N78K,N78M, N78Q, N78R; K80: K80R, K80H, K80S, K80Q, K801, K80E, K80N; E101:E101D, E101H, E101K, E101R, E101S; group 8: K68: K68R, K68H, K68S, K68Q,K681, K68E, K68N; R70: R70K, R70H, R70T, R70D, R70E, R70N; I86: I86L,I86K, I86R, I86D; E87: E87D, E87H, E87K, E87R, E87S, E87A; group 9: G1:G1N, G1H, G1K, G1M, G1Q, G1R; G92: G92N, G92H, G92K, G92M, G92Q, G92R;D93: D93N, D93E, D93S, D93H, D93R, D93K; G124: G124N, G124H, G124K,G124M, G124Q, G124R; H126: H126W, H126F, H126S, H126D; group 10: Y66:Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.
 9. A recombinant Bet v 1 allergenaccording to claim 1 that comprises the following mutations: Y5V, E45S,N78K, K97S, K103V, K134E, +160N.
 10. A recombinant Bet v 1 allergenaccording to claim 9 that further comprises at least one of thefollowing substitutions: E8/K115, D125/H126, E138/K137/E141, D25/N28,E87/K55, S155/H154/N159, N47/P50/H76/N43/144/R70, E87/K55, E73/P50/D72,A130, N28/D25, P108, V2/K119/N4/E6/E96.
 11. A recombinant Bet v 1allergen according to claim 9 that further comprises at least one of thefollowing substitutions: T10P, K65N, N28/D25/K32Q/E141/K137/E138,D125/K123I/H126, P108/D109N, E42S/K55/144/N43, E73/D72, E87, E96/K119,A130, V2/E6, E8/K115, N47/P50/R70/H76/T77A, S155/D156H/N159, E6N2.
 12. Arecombinant Bet v 1 allergen according to claim 10 that furthercomprises at least one of the following substitutions: T10P, K65N,N28/D25/K32Q/E141/K137/E138, D125/K123I/H126, P108/D109N,E42S/K55/144/N43, E73/D72, E87, E96/K119, A130, V2/E6, E8/K115,N47/P50/R70/H76/T77A, S155/D156H/N159, E6N2.
 13. A recombinant Bet v 1allergen according to claim 1 that comprises the following mutations:Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, K134E, +160N.
 14. Arecombinant Bet v 1 allergen according to claim 13 that furthercomprises at least one of the following substitutions: E8/K115,D125/H126, E138/K137/E141, E87/K55, S155/H154/N159,N47/P50/H76/N43/144/R70, K55, E73/P50/D72, A130, D25, P108, V2/K119/N4/E6/E96.
 15. A recombinant Bet v 1 allergen according to claim 13that further comprises at least one of the following substitutions:T10P, K65N, E141/K137/E138, D125/K123I/H126, P108/D109N,E42S/K55/144/N43, E73/D72, E87, V2/E6, N47/P50/R70/H76/T77A. E96/K119,A130, E8/K115, S155/D156H/H154/N159, E6N2.
 16. A recombinant Bet v 1allergen according to claim 14 that further comprises at least one ofthe following substitutions: T10P, K65N, E141/K137/E138,D125/K123I/H126, P108/D109N, E42S/K55/144/N43, E73/D72, E87, V2/E6,N47/P50/R70/H76/T77A. E96/K119, A130, E8/K115, S155/D156H/H154/N159,E6N2.
 17. A recombinant Bet v 1 allergen according to claim 1 thatcomprises the following mutations: Y5V, N28T, K32Q, E45S, N78K, E87S,K97S, K103V, K134E, N159G, +160N.
 18. A recombinant allergen accordingto claim 17, that further comprises at least one of the followingsubstitutions: K55, A138/K137/E141, D125/H126, P108, V2/N4/K119/E6,S155/H154, N47/P50/H76, E73, R70, A130, E8/K115, E96.
 19. A recombinantallergen according to claim 17, that further comprises at least one ofthe following substitutions: K65N, T10P, D125, K123I, P108, D109N,N47/P50/H76, E138/K137/El41, E42S/K55/144/N43, S155/D156H, E73/D72,E6N2, E96.
 20. A recombinant allergen according to claim 18, thatfurther comprises at least one of the following substitutions: K65N,T10P, D125, K123I, P108, D109N, N47/P50/H76, E138/K137/El41,E42S/K55/144/N43, S155/D156H, E73/D72, E6N2, E96.
 21. A recombinant Betv 1 allergen according to claim 1 that comprises the followingmutations: Y5V, N28T, K32Q, E45S, N78K, K97S, K103V, P108G, D125Y,K134E, +160N.
 22. A recombinant Bet v 1 allergen according to claim 21that further comprises at least one of the following substitutions: E87,E141, K55, N47/N43/144/H76, S155/HIS154, A130, E8, E73, V2/K119.
 23. Arecombinant Bet v 1 allergen according to claim 21 that furthercomprises at least one of the following substitutions: K65N, T10P/E8,E87, S155/D156H, E141, E42S, A130, E8/T10P, N47, H76T, V2.
 24. Arecombinant Bet v 1 allergen according to claim 22 that furthercomprises at least one of the following substitutions: K65N, T10P/E8,E87, S155/D156H, E141, E42S, A130, E8/T10P, N47, H76T, V2.
 25. Arecombinant Bet v 1 allergen according to claim 1 that comprises thefollowing mutations: Y5V, N28T, K32Q, E45S, E73S, E96S, P108G, D125Y,N159G, +160N.
 26. A recombinant Bet v 1 allergen according to claim 25that further comprises at least one of the following substitutions:K134, N78, E87, K119, E8, K55X, E141, N47, S155, E6, K103, A130, V2. 27.A recombinant Bet v 1 allergen according to claim 25 that furthercomprises at least one of the following substitutions: K65N/K55,T10P/E8/E141, E138/K134, E87, E42S/K55/144, S155/D156H, N78, K119N2/N4,N47/P50, H76/T77A, A130, E6/K115/K103.
 28. A recombinant Bet v 1allergen according to claim 26 that further comprises at least one ofthe following substitutions: K65N/K55, T10P/E8/E141, E138/K134, E87,E42S/K55/144, S155/D156H, N78, K119N21N4, N47/P50, H76/T77A, A130,E6/K115/K103.
 29. A recombinant Bet v 1 allergen according to claim 1that comprises the following mutations: Y5V, N28T, K32Q, E45S, E96S,P108G, +160N.
 30. A recombinant Bet v 1 allergen according to claim 29that further comprises at least one of the following substitutions:K134, N78, E87, K119, E8, K55X, E141, S155, N47, E6, K103, A130, V2,R70, D125.
 31. A recombinant Bet v 1 allergen according to claim 29 thatfurther comprises at least one of the following substitutions: N78/T77A,K103X, K134/E138, K65N/K55, T10P, D125/H126, E42S/K55,S155/D156H/HIS154, K119N2, E87, N47/P50/H76, A130.
 32. A recombinant Betv 1 allergen according to claim 30 that further comprises at least oneof the following substitutions: N78/T77A, K103X, K134/E138, K65N/K55,T10P, D125/H126, E42S/K55, S155/D156H/HIS154, K119/V2, E87, N47/P50/H76,A130.
 33. A recombinant Bet v 1 allergen according to claim 1 thatcomprises at least one of the following substitutions: Y5, N28, K32,E45, E96/K97, P108/D109, N159/+160, E60, T10, K103/K115, K65, K129,K134, E42/K55, S149/A153/L152, D125/K123, N47/L24, T77/N78, K119, E87,A16/K20/P14, Q36/G61/P63, E73, D93, V2.
 34. A recombinant Bet v 1allergen according to claim 1 that comprises at least one of thefollowing substitutions: Y5V, N28T, K32Q, E45S, E96S/K97S, P108G/D109N,N159G/+160N, E60S, T10N, K103V/K115N, K129N, K134E, E42S/K55N,S149T/A153V/L152A, D125Y/K123I, N47K/L24A, T77N/N78K, K119N, E87A,A116G/K20S/P14G, Q36N/G61S/P63G, E73S, D93S, V2L.
 35. A recombinant Betv 1 mutant allergen according to claim 1 comprising substitutions thatare selected from at least four of the following 10 groups: group 1:A130V, K134E, E141N, group 2: V2L, Y5V, E6S, K 119N, group 3: E42S,E45S, N47K, K55N, E73S, E73T, E73S, group 4: E8S, T10P, P14G, P108G,D109N, K115N, group 5: A16G, K20S, S149T L152A A153V, S155T, N159G,+160N, group 6: L24A, D25E, N28T, K32Q, group 7: T77A, T77N, N78K,K103V, group 8: R70N, E87A, E96S, K97S, group 9: D93S, K1231, D125Y,K129N, group 10: Q36N, E60S, G61S, P63G.
 36. A recombinant Bet v 1mutant allergen according to claim 1 comprising substitutions that areselected from at least four of the following 10 groups: group 1: K134E,group 2: Y5V, K119N, V2L, group 3: E45S, E42S, K55N, N47K, E73S, group4: E96S, K97S, P108G, D109N, T10N, K115N, P14G, group 5: N159G, +160N,S149T, A153V, L152A, A16G, K20S, group 6: N28T, K32Q, L24A, group 7:K103V, T77N, N78K, group 8: E96S, K97S, E87A, group 9: K129N, D125Y,K123I, D93S, group 10: E60S, Q36N, G61S, P63G.
 37. A recombinant Bet v 1allergen according to claim 1 comprising 5-10 primary mutations.
 38. Arecombinant Bet v 1 allergen according claim 1 that further comprises atleast one secondary mutation.
 39. A recombinant Bet v 1 allergenaccording to claim 1 that further comprises at least one secondarymutation selected from the groups listed in claim
 1. 40. A recombinantBet v 1 allergen according to claim 1 that further comprises at leastone additional mutation wherein the mutation is an addition or deletionof a surface exposed loop amino acid residue.
 41. A compositioncomprising two or more recombinant mutant Bet v 1 allergen variantsaccording to claim 1 wherein each variant is defined by having at leastone primary mutation, which is absent in at least one of the othervariants.
 42. A composition according to claim 41 comprising 2-12 ofsaid variants.
 43. A composition according to claim 41 that is apharmaceutical composition.
 44. A composition according to claim 43 thatis suitable for preventing and/or treating Fagales pollen allergy.
 45. Acomposition according to claim 43 that is suitable for preventing and/ortreating birch pollen allergy.
 46. A pharmaceutical compositioncharacterised in that it comprises a recombinant allergen according toclaim 1, optionally in combination with a pharmaceutically acceptablecarrier and/or excipient, and optionally an adjuvant.
 47. Apharmaceutical composition characterised in that it comprises acomposition according to claim 41, optionally in combination with apharmaceutically acceptable carrier and/or excipient, and optionally anadjuvant.
 48. A pharmaceutical composition according to claim 46,characterised in that it is in the form of a vaccine against allergicreactions elicited by a naturally occurring Bet v 1 allergen in patientssuffering from birch pollen allergy.
 49. A pharmaceutical compositionaccording to claim 47, characterised in that it is in the form of avaccine against allergic reactions elicited by a naturally occurring Betv 1 allergen in patients suffering from birch pollen allergy.
 50. Amethod of generating an immune response in a subject comprisingadministering to a subject a recombinant allergen according to claim 1.51. A method of generating an immune response in a subject comprisingadministering to a subject a composition according to claim
 41. 52. Amethod of generating an immune response in a subject comprisingadministering to a subject a pharmaceutical composition according toclaim
 45. 53. Vaccination or treatment of a subject comprisingadministering to the subject a recombinant allergen according toclaim
 1. 54. Vaccination or treatment of a subject comprisingadministering to the subject a composition according to claim
 41. 55.Vaccination or treatment of a subject comprising administering to thesubject a pharmaceutical composition according to claim
 45. 56. Aprocess for preparing a pharmaceutical composition according to claim 45comprising mixing a recombinant allergen or a composition withpharmaceutically acceptable substances and/or excipients.
 57. Apharmaceutical composition obtainable by the process according to claim56.
 58. A method for the treatment, prevention or alleviation ofallergic reactions in a subject comprising administering to a subject arecombinant Bet v 1 allergen according to claim
 1. 59. A method for thetreatment, prevention or alleviation of allergic reactions in a subjectcomprising administering to a subject, a composition according to claim41.
 60. A method for the treatment, prevention or alleviation ofallergic reactions in a subject comprising administering to a subject apharmaceutical composition according to claim
 57. 61. A method ofpreparing a recombinant Bet v 1 allergen according to claim 1 whereinthe substitution of amino acids is carried out by site-directedmutagenesis.
 62. A method of preparing a recombinant Bet v 1 allergenaccording to claim 1, wherein the allergen is produced by DNA shuffling(molecular breeding).
 63. A method of preparing a recombinant Bet v 1allergen library according to claim 1 wherein the allergen is producedby using oligonucleotide primers accommodating random substitutions ofat least four amino residues.
 64. A method according to claim 63 whereinthe amino acid residues are selected from the group consisting of: Y5,T10, K20, N28, K32, Q36, E42, E45, E73, K65, N78, E87, K97, K103, P108,K123, K129, K134, S149, D156, and +160.
 65. A DNA sequence encoding arecombinant Bet v 1 allergen according to claim 1, a derivative thereof,a partial sequence thereof, a degenerated sequence thereof or a sequencewhich hybridises thereto under stringent conditions, wherein saidderivative, partial sequence, degenerated sequence or hybridisingsequence encodes a peptide having at least one B cell epitope.
 66. A DNAsequence according to claim 65, which is a derivative of the DNAsequence encoding the naturally occurring allergen.
 67. A DNA sequenceaccording to claim 66 wherein the derivative is obtained bysite-directed mutagenesis of the DNA encoding the naturally occurringBet v 1 allergen.
 68. An expression vector comprising the DNA accordingto claim
 65. 69. A host cell comprising the expression vector of claim68.
 70. A method of producing a recombinant mutant Bet v 1 allergencomprising the step of cultivating the host cell of claim
 69. 71. Arecombinant Bet v 1 allergen according to claim 1 comprising at leastone T cell epitope capable of stimulating a T cell clone or T cell linespecific for the naturally occurring Bet v 1 allergen.
 72. A recombinantBet v 1 allergen that is encoded by the DNA sequence according to claim65 comprising at least one T cell epitope capable of stimulating a Tcell clone or T cell line specific for the naturally occurring Bet v 1allergen.
 73. A diagnostic assay for assessing relevance, safety, oroutcome of therapy of a subject using a recombinant mutant Bet v 1allergen according to claim 1, wherein an IgE containing sample of asubject is mixed with said mutant and assessed for the level ofreactivity between the IgE in said sample and said mutant.
 74. Adiagnostic assay for assessing relevance, safety, or outcome of therapyof a subject using a composition according to claim 41, wherein an IgEcontaining sample of a subject is mixed with mutant or composition andassessed for the level of reactivity between the IgE in said sample andsaid mutant.